Antibodies against ox-40 and uses thereof

ABSTRACT

Provided herein are antibodies, or antigen binding portions thereof, that bind to OX40. Also provided are uses of these proteins in therapeutic applications, such as in the treatment of cancer. Further provided are cells that produce the antibodies, polynucleotides encoding the heavy and/or light chain variable region of the antibodies, and vectors comprising the polynucleotides encoding the heavy and/or light chain variable region of the antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/847,130, filed Apr. 13, 2020 (currently allowed), which is acontinuation application of U.S. application Ser. No. 15/474,731, filedMar. 30, 2017 (now issued as U.S. Pat. No. 10,683,357), which is adivisional application of U.S. application Ser. No. 15/166,114, filedMay 26, 2016 (now issued as U.S. Pat. No. 9,644,032), which claimspriority to U.S. Provisional Application Nos. 62/333,556, 62/327,140,62/264,691, 62/239,574, and 62/168,377, filed May 9, 2016, Apr. 25,2016, Dec. 8, 2015, Oct. 9, 2015, and May 29, 2015, respectively. Thecontents of the aforementioned applications are hereby incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 11, 2022, isnamed 3338_1140009_Seqlisting_ST25.txt and is 417,546 bytes in size.

BACKGROUND

OX40 (TNFRSF4), also known as ACT35, IMD16, TXGP1L, and CD134, is a50-kD type I transmembrane glycoprotein in the TNFSFR family ofcostimulatory receptors expressed on activated CD4+ T cells. In thecontext of cancer, OX40-expressing activated T cells are found in tumorinfiltrating lymphocytes. OX40 and its ligand, OX40-L, play a crucialrole in inducing and maintaining T-cell responses. Recent studies havedemonstrated the utility of enhancing anti-tumor T cell function tofight cancer, with key components of an effective response including theactivation of CD4+ T cells and promoting survival signals through memoryand effector T cells. Given the ongoing need for improved strategies fortreating diseases such as cancer through, e.g., enhancing immuneresponses such as T cell responses, novel agents that modulate T cellresponses, such as those that target OX40, as well as therapies (e.g.,combination therapies) that use such agents, would be therapeuticallybeneficial.

SUMMARY

Provided herein are antibodies, such as human monoclonal antibodies,that specifically bind OX40 and have desirable functional properties.These properties include high affinity binding to human OX40 andcynomolgus OX40 and the ability to stimulate antigen-specific T cellresponses, e.g., in tumor-bearing subjects. Also provided herein aremethods of detecting OX40 in a sample.

In one aspect, provided herein are antibodies, or antigen-bindingportions thereof, which specifically bind to OX40 and exhibit at leastone of the following properties:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

In certain embodiments, the anti-OX40 antibodies, or antigen bindingportions thereof, described herein stimulate an anti-tumor immuneresponse, for example, an antigen-specific T cell response. In certainembodiments, the antibodies, or antigen binding portions thereof,increase cytokine production (e.g., IL-2 and/or IFN-γ) inOX40-expressing T cells and/or increase T cell proliferation. In certainembodiments, the antibodies bind to the C1q component of humancomplement. In certain embodiments, the antibodies induce NKcell-mediated lysis of activated CD4+ T cells. In certain embodiments,the antibody promotes macrophage-mediated phagocytosis of OX40expressing cells. In certain embodiments, the antibody inhibitsregulatory T cell-mediated suppression of CD4+ T cell proliferation.

In certain embodiments, the anti-OX40 antibodies, or antigen bindingportions thereof, bind to Fc receptors, such as one or more activatingFcγRs. In certain embodiments, the antibodies, or antigen bindingportions thereof, induce or enhance T cell activation throughmultivalent crosslinking.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which specifically bind to OX40 and comprise the threevariable heavy chain CDRs and the three variable light chain CDRs thatare in the variable heavy chain and variable light chain pairs selectedfrom: SEQ ID NOs: 318 and 94; SEQ ID NOs: 17 and 18; 28 and 29; 28 and30; 37 and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and 74; 84and 85; 84 and 86; 93 and 94.

Also provided herein are monoclonal antibodies, or antigen bindingportions thereof, which bind to OX40 and comprise:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87, 317, and 89, respectively, and light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:11-13, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 14-16, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 22-24, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 25-27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:31-33, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 34-36, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 42-44, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 45-47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:51-53, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 54-56, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:59-61, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 62-64, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:67-69, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 70-72, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 78-80, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 81-83, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87-89, respectively, and light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 90-92, respectively.

Provided herein are monoclonal antibodies, or antigen binding portionsthereof, which bind to OX40 and comprise heavy and light chain variableregions, wherein the heavy chain variable region comprises an amino acidsequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence selected from the group consistingof SEQ ID NOs: 318, 17, 28, 37, 48, 57, 65, 73, 84, and 93.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to OX40 and comprise heavy and light chainvariable regions, wherein the light chain variable region comprises anamino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 94, 18, 29, 30, 38, 49, 50, 58, 66, 74, 85,86, and 94.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to OX40 and comprise heavy and light chainvariable region sequences at least 85% identical, for example, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical, to the amino acid sequencesselected from the group consisting of: SEQ ID NOs: 318 and 94; 17 and18; 28 and 29; 28 and 30; 37 and 38; 48 and 49; 48 and 50; 57 and 58; 65and 66; 73 and 74; 84 and 85; 84 and 86; 93 and 94.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to OX40 and comprise heavy chain and lightchain sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100%identical to the amino acid sequences selected from the group consistingof: SEQ ID NOs: 124 and 116; 95 and 96; 97 and 98; 99 and 100; 101 and102; 103 and 104; 105 and 106; 107 and 108; 109 and 110; 111 and 112;113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and 122; 123 and116; 124 and 116; and 125 and 116.

In certain embodiments, the isolated monoclonal antibodies, or antigenbinding portions thereof, (a) bind to the same epitope on OX40 as 3F4,14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3, or 20C1, or binds to the sameepitope on OX40 as 6E1-1, 6E1-2, 14A2-1 or 14A2-2, and (b) inhibitbinding of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,14A2-1, 14A2-2, and/or 20C1 to OX40 on activated T cells by at least50%, 60%, 70%, 80% or 90% as measured by, e.g., FACS.

In certain embodiments, the anti-OX40 antibodies, or antigen bindingportions thereof, bind within the regions DVVSSKPCKPCTWCNLR (SEQ ID NO:178) or DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of matureextracellular portion of human OX40 (SEQ ID NO: 2). In some embodiments,the anti-OX40 antibodies, or antigen binding portions thereof, describedherein, bind to both human and cynomolgus OX40. In some embodiments, theanti-OX40 antibodies, or antigen binding portions thereof, describedherein, do not bind to mouse and/or rat OX40.

In certain embodiments, the anti-OX40 antibodies, or antigen-bindingportions thereof, are IgG1, IgG2, IgG3, or IgG4 antibodies, or variantsthereof. In certain embodiments, methionine residues in the CDR regionsof the anti-OX40 antibodies, or antigen-binding portions thereof, aresubstituted for amino acid residues that do not undergo oxidation. Incertain embodiments, the anti-OX40 antibodies, or antigen-bindingportions thereof, are human or humanized antibodies. In certainembodiments, the anti-OX40 antibodies comprise an Fc having enhancedbinding to an activating FcγR.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to OX40 comprising a modified heavy chainconstant region that comprises an IgG2 hinge and at least one of CH1,CH2 and CH3 that is not of an IgG2 isotype.

In certain embodiments, the heavy chain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125, or aheavy chain that differs therefrom in at most 10 amino acids or is atleast 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence ofSEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 124 and 125.

In certain embodiments, the light chain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, and 122, or a light chain thatdiffers therefrom in at most 10 amino acids or is at least 95%, 96%,97%, 98% or 99% identical to an amino acid sequence of SEQ ID NOs: 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.

In certain embodiments, the anti-OX40 antibodies, or antigen-bindingportions thereof, are not immunogenic.

In certain embodiments, the anti-OX40 antibodies, or antigen-bindingportions thereof, lack an amino acid sequence that undergoesisomerization. For instance, if the amino acid sequence Asp-Gly ispresent in the heavy and/or light chain CDR sequences of the antibody,the sequence is substituted with an amino acid sequence that does notundergo isomerization. In one embodiment, the antibody comprises theheavy chain variable region CDR2 sequence set forth in SEQ ID NO: 76,but wherein the Asp or Gly in the Asp-Gly sequence (LISYDGSRKHYADSVKG;SEQ ID NO: 76) is replaced with an amino acid sequence that does notundergo isomerization, for example, an Asp-Ser or a Ser-Gly sequence. Inanother embodiment, the antibody comprises the heavy chain variableregion CDR2 sequence set forth in SEQ ID NO: 88, but wherein the Asp orGly in the Asp-Gly sequence (AIDTDGGTFYADSVRG; SEQ ID NO: 88) isreplaced with an amino acid sequence that does not undergoisomerization, for example, a Ser-Gly, an Asp-Ala, or a Ser-Thrsequence.

Provided herein are antibodies which bind to OX40 comprising an aminoacid selected from the group consisting of SEQ ID NOs: 282-296. In oneembodiment, the antibodies comprise a heavy chain consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 282-296.

Provided herein are bispecific molecules comprising an anti-OX40antibody linked to a molecule having a second binding specificity.

Provided herein are nucleic acids encoding the heavy and/or light chainvariable regions of the anti-OX40 antibodies, or antigen bindingportions thereof, expression vectors comprising the nucleic acidmolecules, and cells transformed with the expression vectors.

Provided herein are immunoconjugates comprising the anti-OX40 antibodiesdescribed herein, linked to an agent.

Provided herein are compositions comprising anti-OX40 antibodies, orantigen binding portions thereof, and a carrier.

Provided herein are kits comprising the anti-OX40 antibodies, or antigenbinding portions thereof, and instructions for use. In certainembodiments, the kits further comprise an anti-CTLA4 antibody,anti-PD-1, or anti-PD-L1 antibody.

Provided herein is a method of preparing the anti-OX40 antibodies,comprising expressing an anti-OX40 antibody in a cell and isolating theantibody from the cell.

Provided herein is a method of stimulating an antigen-specific T cellresponse comprising contacting the T cell with an anti-OX40 antibody, orantigen binding portion thereof, such that an antigen-specific T cellresponse is stimulated.

Provided herein is a method of activating or co-stimulating a T cell,e.g., an effector T cell, comprising contacting a cell, e.g., aneffector T cell, with an anti-OX40 antibody, or antigen binding portionthereof, and CD3, wherein the effector T cell is activated orco-stimulated.

Provided herein is a method of increasing IL-2 and/or IFN-γ productionin and/or proliferation of a T cell comprising contacting the T cellwith an effective amount of an anti-OX40 antibody, or antigen bindingportion thereof.

Provided herein is a method of increasing IL-2 and/or IFN-γ productionin T cells in a subject comprising administering an effective amount ofan anti-OX40 antibody, or antigen binding portion thereof, bispecificmolecule or conjugate comprising the anti-OX40 antibody, or compositioncomprising the anti-OX40 antibody, to increase IL-2 and/or IFN-γproduction from the T cells.

Provided herein is a method of reducing or depleting the number of Tregulatory cells in a tumor of a subject in need thereof comprisingadministering an effective amount of an anti-OX40 antibody, or antigenbinding portion thereof, bispecific molecule or conjugate wherein theantibody, or antigen binding portion thereof, has effector or enhancedeffector function, to reduce the number of T regulatory cells in thetumor.

Provided herein is a method of stimulating an immune response in asubject comprising administering an effective amount of an anti-OX40antibody, or antigen binding portion thereof, bispecific molecule orconjugate to the subject such that an immune response in the subject isstimulated. In certain embodiments, the subject has a tumor and animmune response against the tumor is stimulated.

Provided herein is a method of inhibiting the growth of tumor cells in asubject comprising administering to the subject an anti-OX40 antibody,or antigen binding portion thereof, bispecific molecule or conjugatesuch that growth of the tumor is inhibited in the subject.

Provided herein is a method of treating cancer, e.g., by immunotherapy,comprising administering to a subject in need thereof a therapeuticallyeffective amount an anti-OX40 antibody, or antigen binding portionthereof, bispecific molecule or conjugate comprising the anti-OX40antibody, or composition comprising the anti-OX40 antibody, to treat thecancer. In certain embodiments, the cancer is selected from the groupconsisting of: bladder cancer, breast cancer, uterine/cervical cancer,ovarian cancer, prostate cancer, testicular cancer, esophageal cancer,gastrointestinal cancer, pancreatic cancer, colorectal cancer, coloncancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer. In certain embodiments, thecancer is a metastatic cancer, refractory cancer, or recurrent cancer.

In certain embodiments, the methods described herein further compriseadministering one or more additional therapeutics with an anti-OX40antibody, for example, an anti-PD1 antibody, a LAG-3 antibody, a CTLA-4antibody, and/or a PD-L1 antibody.

Provided herein is a method of detecting the presence of OX40 in asample comprising contacting the sample with an anti-OX40 antibody, oran antigen binding portion thereof, under conditions that allow forformation of a complex between the antibody, or antigen binding portionthereof, and OX40, and detecting the formation of a complex.

Provided herein are uses of the anti-OX40 antibodies described hereinfor treating cancer, stimulating an immune response in a subject,stimulating an antigen-specific T cell response, activating orco-stimulating a T cell, increasing the production of cytokines, such asIL-2 and/or IFN-γ, in and/or proliferation of a T cell, reducing ordepleting the number of T regulatory cells in a tumor, and/or inhibitingthe growth of tumor cells. Also provided herein are uses of theanti-OX40 antibodies described herein for preparing a medicament forstimulating an immune response in a subject, stimulating anantigen-specific T cell response, activating or co-stimulating a T cell,increasing IL-2 and/or IFN-γ production in and/or proliferation of a Tcell, reducing or depleting the number of T regulatory cells in a tumor,and/or inhibiting the growth of tumor cells.

Provided herein is a method of treating a solid tumor in a humansubject, the method comprising administering to the subject an effectiveamount of an anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domainsof the heavy chain variable region having the sequence set forth in SEQID NO: 318, and CDR1, CDR2 and CDR3 domains of the light chain variableregion having the sequence set forth in SEQ ID NO: 94, wherein themethod comprises at least one administration cycle, wherein the cycle isa period of two weeks, wherein for each of the at least one cycles, onedose of the anti-OX40 antibody is administered at a dose of 1 mg/kg bodyweight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about 1mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or 320 mg.

In one embodiment, the method comprises further administering ananti-PD-1 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 301,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 302, wherein the methodcomprises at least one administration cycle, wherein the cycle is aperiod of two, three, or four weeks, wherein for each of the at leastone cycles, one dose of the anti-OX40 antibody is administered at a doseof 1 mg/kg body weight; a fixed dose of 20, 40, 80, 160, or 320 mg; adose of about 1 mg/kg body weight; or a fixed dose of about 20, 40, 80,160, or 320 mg, and one dose of the anti-PD-1 antibody is administeredat a dose of 240, 360, or 480 mg or a dose of about 240, 360, or 480 mg.

In another embodiment, the method comprises further administering ananti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 309,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 310, wherein the methodcomprises at least one administration cycle, wherein the cycle is aperiod of three weeks, wherein for each of the at least one cycles, onedose of the anti-OX40 antibody is administered at a dose of 1 mg/kg bodyweight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about 1mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or 320 mg,and one dose of the anti-CTLA-4 antibody is administered at a dose of 1mg/kg or a dose of about 1 mg/kg,

wherein the anti-OX40 antibody is administered together with theanti-CTLA-4 antibody for at least one cycle, followed by anti-OX40antibody monotherapy for at least one cycle. In some embodiments, thetreatment consists of 8 cycles. In one embodiment, the anti-OX40antibody is administered together with the anti-CTLA-4 antibody for thefirst 4 cycles, followed by anti-OX40 antibody monotherapy for the last4 cycles.

In some embodiments, the anti-OX40 antibody comprises heavy and lightchain sequences set forth in SEQ ID NOs: 124 and 116, respectively.

In certain embodiments, the anti-OX40 antibody, or anti-OX40 antibodyand anti-PD-1 or anti-CTLA-4 antibody, are formulated for intravenousadministration. In some embodiments, the anti-OX40 and anti-PD-1 oranti-CTLA-4 antibody are formulated together. In other embodiments, theanti-OX40 and anti-PD-1 or anti-CTLA-4 antibody are formulatedseparately.

In some embodiments, the treatment consists of 8 cycles. In oneembodiment, the anti-OX40 antibody, or anti-OX40 antibody and anti-PD-1or anti-CTLA-4 antibody, are administered on Day 1 of each cycle.

In certain embodiments, the anti-OX40 antibody is administered prior toadministration of the anti-PD-1 or anti-CTLA-4 antibody. In oneembodiment, the anti-OX40 antibody is administered within about 30minutes prior to administration of the anti-PD-1 or anti-CTLA-4antibody. In other embodiments, the anti-OX40 antibody is administeredafter administration of the anti-PD-1 or anti-CTLA-4 antibody. In yetother embodiments, the anti-OX40 antibody is administered concurrentlywith the anti-PD-1 or anti-CTLA-4 antibody.

In certain embodiments, the treatment produces at least one therapeuticeffect chosen from a reduction in size of a tumor, reduction in numberof metastatic lesions over time, complete response, partial response,and stable disease. In some embodiments, the tumor is associated with acancer selected from the group consisting of: cervical cancer, bladdercancer, colorectal cancer, and ovarian cancer.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 126) and amino acidsequence (SEQ ID NO: 17) of the heavy chain variable region of the 3F4human monoclonal antibody. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO:12) and CDR3 (SEQ ID NO: 13) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 127) and amino acidsequence (SEQ ID NO: 18) of the kappa light chain variable region of the3F4 human monoclonal antibody. The CDR1 (SEQ ID NO: 14), CDR2 (SEQ IDNO: 15) and CDR3 (SEQ ID NO: 16) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 128) and amino acidsequence (SEQ ID NO: 28) of the heavy chain variable region of the 14B6(14B6-1 and 14B6-2) human monoclonal antibody. The CDR1 (SEQ ID NO: 19),CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO: 21) regions are delineated andthe V, D and J germline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 129) and amino acidsequence (SEQ ID NO: 29) of the kappa light chain variable region of the14B6-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 22), CDR2 (SEQ IDNO: 23) and CDR3 (SEQ ID NO: 24) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2C shows the nucleotide sequence (SEQ ID NO: 130) and amino acidsequence (SEQ ID NO: 30) of the kappa light chain variable region of the14B6-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 26), CDR2 (SEQ IDNO: 27) and CDR3 (SEQ ID NO: 28) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 131) and amino acidsequence (SEQ ID NO: 37) of the heavy chain variable region of the 23H3human monoclonal antibody. The CDR1 (SEQ ID NO: 31), CDR2 (SEQ ID NO:32) and CDR3 (SEQ ID NO: 33) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 132) and amino acidsequence (SEQ ID NO: 38) of the kappa light chain variable region of the23H3 human monoclonal antibody. The CDR1 (SEQ ID NO: 34), CDR2 (SEQ IDNO: 35) and CDR3 (SEQ ID NO: 36) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 133) and amino acidsequence (SEQ ID NO: 48) of the heavy chain variable region of the 6E1(6E1-1 and 6E1-2) human monoclonal antibody. The CDR1 (SEQ ID NO: 39),CDR2 (SEQ ID NO: 40) and CDR3 (SEQ ID NO: 41) regions are delineated andthe V, D and J germline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 134) and amino acidsequence (SEQ ID NO: 49) of the kappa light chain variable region of the6E1-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 42), CDR2 (SEQ IDNO: 43) and CDR3 (SEQ ID NO: 44) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4C shows the nucleotide sequence (SEQ ID NO: 135) and amino acidsequence (SEQ ID NO: 50) of the kappa light chain variable region of the6E1-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 45), CDR2 (SEQ IDNO: 46) and CDR3 (SEQ ID NO: 47) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 136) and amino acidsequence (SEQ ID NO: 57) of the heavy chain variable region of the 18E9human monoclonal antibody. The CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO:52) and CDR3 (SEQ ID NO: 53) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 137) and amino acidsequence (SEQ ID NO: 58) of the kappa light chain variable region of the18E9 human monoclonal antibody. The CDR1 (SEQ ID NO: 54), CDR2 (SEQ IDNO: 55) and CDR3 (SEQ ID NO: 56) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 138) and amino acidsequence (SEQ ID NO: 65) of the heavy chain variable region of the 8B11human monoclonal antibody. The CDR1 (SEQ ID NO: 59), CDR2 (SEQ ID NO:60) and CDR3 (SEQ ID NO: 61) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 139) and amino acidsequence (SEQ ID NO: 66) of the kappa light chain variable region of the8B11 human monoclonal antibody. The CDR1 (SEQ ID NO: 62), CDR2 (SEQ IDNO: 63) and CDR3 (SEQ ID NO: 64) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 140) and amino acidsequence (SEQ ID NO: 73) of the heavy chain variable region of the 20B3human monoclonal antibody. The CDR1 (SEQ ID NO: 67), CDR2 (SEQ ID NO:68) and CDR3 (SEQ ID NO: 69) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO: 141) and amino acidsequence (SEQ ID NO: 74) of the kappa light chain variable region of the20B3 human monoclonal antibody. The CDR1 (SEQ ID NO: 70), CDR2 (SEQ IDNO: 71) and CDR3 (SEQ ID NO: 72) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 8A shows the nucleotide sequence (SEQ ID NO: 142) and amino acidsequence (SEQ ID NO: 84) of the heavy chain variable region of the 14A2(14A2-1 and 14A2-2) human monoclonal antibody. The CDR1 (SEQ ID NO: 75),CDR2 (SEQ ID NO: 76) and CDR3 (SEQ ID NO: 77) regions are delineated andthe V, D and J germline derivations are indicated.

FIG. 8B shows the nucleotide sequence (SEQ ID NO: 143) and amino acidsequence (SEQ ID NO: 85) of the kappa light chain variable region of the14A2-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 78), CDR2 (SEQ IDNO: 79) and CDR3 (SEQ ID NO: 80) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 8C shows the nucleotide sequence (SEQ ID NO: 144) and amino acidsequence (SEQ ID NO: 86) of the kappa light chain variable region of the14A2-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 81), CDR2 (SEQ IDNO: 82) and CDR3 (SEQ ID NO: 83) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 9A shows the nucleotide sequence (SEQ ID NO: 145) and amino acidsequence (SEQ ID NO: 93) of the heavy chain variable region of the 20C1human monoclonal antibody. The CDR1 (SEQ ID NO: 87), CDR2 (SEQ ID NO:88) and CDR3 (SEQ ID NO: 89) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 9B shows the nucleotide sequence (SEQ ID NO: 146) and amino acidsequence (SEQ ID NO: 94) of the kappa light chain variable region of the20C1 human monoclonal antibody. The CDR1 (SEQ ID NO: 90), CDR2 (SEQ IDNO: 91) and CDR3 (SEQ ID NO: 92) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 10A shows the nucleotide sequence (SEQ ID NO: 176) and amino acidsequence (SEQ ID NO: 124) of the heavy chain of the OX40.21 humanmonoclonal antibody. The nucleotide sequence (SEQ ID NO: 168) and aminoacid sequence (SEQ ID NO: 116) of the light chain is shown in FIG. 10B.

FIGS. 11A, 11B, 11C, and 11D show binding curves and EC₅₀s (in nM) ofvarious anti-OX40 antibodies for activated human T cells, with hIgG1 andsecondary antibodies serving as controls, as assessed by FACS.

FIGS. 12A, 12B, and 12C show binding curves and EC₅₀s (in nM) of variousanti-OX40 antibodies for activated cynomolgus T cells, with hIgG1 andsecondary antibodies serving as controls, as assessed by FACS.

FIGS. 13A, 13B, and 13C show binding curves and K_(D)s of the anti-OX40antibody, OX40.21, for activated human T cells, HEK293 cellsoverexpressing human OX40, and CHO cells overexpressing cynomolgusmonkey OX40, as assessed by Scatchard analysis.

FIG. 14A shows immunohistological staining of various acetone-fixedfrozen human tissue sections with the anti-OX40 antibodies OX40.8,OX40.6, and OX40.16. Images show representative staining at an antibodyconcentration of 1 μg/ml for hyperplasic tonsil and 5 μg/ml for othertissues, with the exception of OX40.16 at 10 μg/ml in the endocardiumand valves. While all three antibodies positively stained a small subsetof lymphocytes in the tonsil, OX40.8 also stained myofilament-likestructures in the heart and OX40.6 stained the cardiac muscles,endothelium/subendothelium matrix in small arteries in the tonsil, andendocardium and valves in the heart. GC, germinal center; MZ, mantlezone.

FIG. 14B shows immunostaining of various acetone-fixed frozen humantissue sections with the anti-OX40 antibody OX40.21 (a variant ofOX40.16). Images show representative staining at an antibodyconcentration of 5 μg/ml. The antibody positively stained a small subsetof lymphocytes in the tonsil and thymus. The positive cells in thetonsil were distributed in the germinal center, mantle zone, andinter-follicular region, while the positive cells in the thymus wereprimarily localized in the medulla. No specific staining was observed inthe heart, liver, kidney, and lung. GC, germinal center; Me, medulla;MZ, mantle zone.

FIG. 15A shows the distribution of OX40+ tumor infiltrating lymphocytesin hepatocellular carcinoma (HCC), colorectal carcinoma (CRC), head andneck squamous cell carcinoma (HNSCC), and melanoma (Mel). A manual scoreof 12 to 20 cases for each tumor type was performed by estimation ofnumber of positive cells under the 20× objective of a microscope.Minimum, <1 cells per 20× objective field; Mild, 1˜<10 cells per 20×objective field; Moderate, 10˜<50 cells per 20× objective field; Marked,50˜<200 cells per 20× objective; Intense, >200 cells per 20× objectivefield.

FIGS. 15B and 15C shows immunohistological staining for CD3, FoxP3, andOX40 on adjacent FFPE sections from colorectal carcinoma (CRC) and headand neck squamous cell carcinoma (HNSCC) samples. FIG. 15B is a lowpower view, showing that both OX40+ and FoxP3+ TILs are a small fractionof CD3+ TILs and primarily distributed in tumor stroma. FIG. 15C is ahigher power view showing potential partial co-localization of OX40+ andFoxP3+(Treg) TILs in CRC.

FIG. 16 shows the ability of various anti-OX40 antibodies to inhibit thebinding of OX40 ligand (OX40-L) to human OX40-transfected 293 cells(“hOX40-293 cells”), with hIgG1 as the control.

FIG. 17 summarizes the OX40-L blocking relationships between variousanti-OX40 antibodies.

FIG. 18 shows the ability of various anti-OX40 antibodies to inhibit thebinding of the anti-OX40 antibody clone L106 to hOX40-293 cells, asassessed by FACS, with PE-labeled L106 only, PE-labeled mIgG1, andunstained cells as controls.

FIGS. 19A, 19B, and 19C show the ability of various anti-OX40 antibodiesto inhibit the binding of allophycocyanin (APC)-conjugated OX40.1antibody to hOX40-293 (FIGS. 19A and 19B) or hOX40-HT1080 cells (FIG.19C), as assessed by FACS. hIgG1 and/or hIgG4 were used as controls.

FIGS. 19D, 19E, 19F, and 19G show the ability of various anti-OX40antibodies to inhibit the binding of biotin-conjugated OX40.4 or OX40.5antibody to hOX40-293 cells. hIgG1 and streptavidin-APC were used ascontrols.

FIG. 19H summarize the epitope bins in relation to OX40.1 based onresults shown in FIGS. 19A, 19B, and 19C.

FIG. 19I summarizes the epitope bins in relation to OX40.5 or OX40.4based on results shown in FIGS. 19D, 19E, 19F, and 19G.

FIG. 20A shows that OX40.21 binds only to non-reduced human OX40,regardless of the presence of N-linked sugars.

FIGS. 20B and 20C show two N-glycopeptides that were identified bypeptide mapping after deglycosylation: 60% occupancy for both AspN118(FIG. 20B) and AspN12 (FIG. 20C).

FIG. 20D depicts the regions in OX40 bound by OX40.16, OX40.21, andOX40.8.

FIG. 20E shows the identification of the peptides recognized by OX40.16,OX40.21, and OX40.8 by LC-MS.

FIGS. 21A, 21B, 21C, and 21D show the effects of various anti-OX40antibodies on human primary CD4+ T cell proliferation when co-culturedwith CHO-CD3-CD32A cells. CHO cells only, T cells only, CHO cellsco-cultured with T cells only, and hIgG1 were used as controls.

FIGS. 22A, 22B, 22C, and 22D show the effects of various anti-OX40antibodies on interferon gamma (IFN-γ) secretion from human primary CD4+T cells co-cultured with CHO-CD3-CD32A cells. CHO cells only, T cellsonly, CHO cells co-cultured with T cells only, and hIgG1 were used ascontrols.

FIGS. 23A, 23B, 23C, 23D, 23E, and 23F show the effects of variousanti-OX40 antibodies on the stimulation of IL-2 secretion from primary Tcells in cultures of staphylococcus enterotoxin B (SEB)-activated humanperipheral blood mononuclear cells (PBMCs), which were isolated fromdifferent donors.

FIG. 24 shows the effects of various anti-OX40 antibodies on NK92 cellinduced lysis of activated CD4+ cells.

FIGS. 25A and 25B show the effects of various anti-OX40 antibodies onprimary NK cell-mediated lysis of activated CD4+ T cells isolated fromtwo donors by NK:target cell ratios.

FIG. 26 shows the effects of various anti-OX40 antibodies on thephagocytosis of hOX40-293 cells by primary human macrophages.

FIG. 27 shows the level of binding of the human complement C1q componentto OX40.21. IgG1 and IgG1.1 (effectorless) were used as controls.

FIGS. 28A, 28B, and 28C show that OX40 is expressed in tumorinfiltrating lymphocytes, with a pattern generally limited to CD4+ cellswith minimal expression on CD8+ cells.

FIG. 28D shows that OX40 is expressed by CD4+ T cells and regulatory Tcells in mouse Sa1N tumors.

FIG. 28E shows that OX40 is expressed by CD4+ T cells, CD8+ T cells, andregulatory T cells in mouse MC38 tumors.

FIG. 29 shows the ability of various anti-OX40 antibodies to reverseregulatory T (Treg) cell-mediated suppression of human CD4+ T cells. Inboth the presence and absence of Treg cells, anti-OX40 antibodiesincreased the proliferation of T responder (Tresp) cells compared to theIgG1 isotype control.

FIGS. 30A and 30B show the clearance of intravenously administeredOX40.6 antibody from monkeys. Two of the monkeys showed acceleratedclearance, which correlated with the formation of anti-drug antibodies.

FIG. 31 is a graph depicting T-cell proliferation results for percentageantigenicity for various anti-OX40 antibodies, as well as qualitycontrol samples QC-1, QC-2, and QC-3.

FIGS. 32A, 32B, and 32C show the effects of different isotypes of thechimeric OX86 antibody (an antibody having the rat variable regions ofOX86 and mouse constant region that does not block the interactionbetween OX40 and OX40-L, i.e., a non-blocking antibody) on anti-tumoractivity measured by changes in tumor volumes in individual mice treatedwith these isotypes (mIgG1 and mIgG2 isotypes) in a MC38 colonadenocarcinoma model: (FIG. 32A) control mouse IgG1 antibody(“control”), (FIG. 32B) OX86 mIgG1 antibody (“OX-40 mIgG1”), (FIG. 32C)OX86 mIgG2 antibody (“OX-40 mIgG2a).

FIGS. 33A, 33B, and 33C show the effects of different isotypes (mIgG1and mIgG2a) of the OX86 antibody on the number of CD4+ regulatory Tcells in tumors and the periphery, and on cell numbers in the spleen.

FIGS. 34A, 34B, and 34C show the effects of chimeric OX86 antibodieswith a human IgG1 on anti-tumor activity measured by changes in tumorvolumes in individual mice treated with the indicated antibodies in theMC38 colon adenocarcinoma model: (FIG. 34A) human IgG1 isotype control(“Isotype”), (FIG. 34B) OX86-hIgG1 chimeric antibody (“OX86-hG1”), (FIG.34C) OX86-hIgG1-S267E antibody (“OX86-hG1-S267E”). The S267Esubstitution in hIgG1 increases its effector function by increasingbinding to FcRs (CD32A and CD32B). OX86-hIgG1 exhibited potentanti-tumor activity.

FIGS. 35A, 35B, 35C, and 35D show the effects of chimeric OX86 hIgG1antibody on regulatory T cell depletion.

FIGS. 36A, 36B, 36C, 36D, and 36E show the effects of a blocking (i.e.,blocks the interaction between OX40 and OX40 ligand) hamster anti-mouseOX40 antibody (8E5) at different dosages on anti-tumor activity bychanges in tumor volumes in individual mice treated with the indicatedantibodies in the subcutaneous mouse CT-26 tumor model. (FIG. 36A)hamster Ig control, (FIG. 36B) 8E5 at 10 mg/kg, (FIG. 36C) 8E5 at 3mg/kg, (FIG. 36D) 8E5 at 1 mg/kg, (FIG. 36E) 8E5 at 0.3 mg/kg.

FIGS. 37A, 37B, 37C, and 37D show the effects of combination therapywith the OX86-rG1 antibody and an anti-PD1 antibody on anti-tumoractivity measured by changes in tumor volumes in individual mice treatedwith the indicated antibodies and combination in the MC38 colonadenocarcinoma model: (FIG. 37A) isotype control, (FIG. 37B) anti-PD1antibody, (FIG. 37C) OX86-rG1 (“anti-OX40”), (FIG. 36D) OX86-rG1+anti-PD1 antibody (“anti-OX40+ anti-PD1”).

FIGS. 38A and 38B show the ex vivo recall response to KLH, CD69+expressing CD4+CD8− T cells at Days 22 and 41, respectively. Animalswere immunized with KLH on Study Day 1. Data points represent individualanimal (males and females) results as a percentage of CD4+CD8− T cells.Horizontal bars represent group means.

FIG. 39 shows antibody binding to anti-his Fab captured FcγR-hisproteins. Binding responses are plotted as a percentage of thetheoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. The barsfor each antibody are shown in the order provided by the color legendsat the bottom of the slide.

FIG. 40 shows antibody binding to anti-his Fab captured FcgR-hisproteins. Binding responses are plotted as a percentage of thetheoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. The barsfor each antibody are shown in the order provided by the color legendsat the bottom of the slide.

FIGS. 41A, 41B, 41C, and 41D show the effects of combination therapywith an agonistic anti-OX40 antibody (OX86-rG1) and an anti-CTLA-4antibody (9D9-mG2b) on anti-tumor activity measured by changes in tumorvolumes in individual mice treated with the indicated antibodies andcombination in the CT26 colon adenocarcinoma model: (FIG. 41A) isotypecontrol, (FIG. 41B) anti-CTLA-4 antibody, (FIG. 41C) OX86-rG1, (FIG.41D) OX86-rG1+ anti-CTLA-4 antibody.

FIG. 42 is a schematic of the Phase 1/2a clinical trial protocol.

FIG. 43 is a schematic of the study visit schedule for the Phase 1/2aclinical trial.

DETAILED DESCRIPTION

Described herein are isolated antibodies, particularly monoclonalantibodies, e.g., human monoclonal antibodies, which specifically bindto OX40 and thereby activate downstream OX40 signaling (“agonistanti-OX40 antibodies”). In certain embodiments, the antibodies describedherein are characterized by particular functional features and/orcomprise particular structural features, such as CDR regions comprisingspecific amino acid sequences. Provided herein are isolated antibodies,methods of making such anti-OX40 antibodies, immunoconjugates, andbispecific molecules comprising such antibodies, and pharmaceuticalcompositions formulated to contain the antibodies. Also provided hereinare methods of using the antibodies for immune response enhancement,alone or in combination with other immunostimulatory agents (e.g.,antibodies) and/or cancer therapies. Accordingly, the anti-OX40antibodies described herein may be used in a treatment in a wide varietyof therapeutic applications, including, for example, inhibiting tumorgrowth and treating viral infections.

Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “OX40” as used herein refers to a receptor that is a member ofthe TNF-receptor superfamily, which binds to OX40 ligand (OX40-L). OX40is also referred to as tumor necrosis factor receptor superfamily,member 4 (TNFRSF4), ACT35, IMD16, TXGP1L, and CD134. The term “OX40”includes any variants or isoforms of OX40 which are naturally expressedby cells. Accordingly, antibodies described herein may cross-react withOX40 from species other than human (e.g., cynomolgus OX40).Alternatively, the antibodies may be specific for human OX40 and may notexhibit any cross-reactivity with other species. OX40 or any variantsand isoforms thereof, may either be isolated from cells or tissues whichnaturally express them or be recombinantly produced using well-knowntechniques in the art and/or those described herein.

The amino acid sequence of human OX40 precursor (Accession No.NP_003318.1) is set forth in SEQ ID NO: 1. The amino acid sequence ofthe extracellular domain of mature human OX40 is set forth in SEQ ID NO:2. The amino acid sequence of cynomolgus OX40 is set forth in SEQ ID NO:3. The amino acid sequence of human OX40-L is set forth in SEQ ID NO: 4.

The terms “Programmed Death 1,” “Programmed Cell Death 1,” “ProteinPD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-I,” as used herein, areused interchangeably, and include variants, isoforms, species homologsof human PD-1, and analogs having at least one common epitope with PD-1.The complete PD-1 sequence can be found under GenBank Accession No.U64863.

The term “cytotoxic T lymphocyte-associated antigen-4,” “CTLA-4,”“CTLA4,” “CTLA-4 antigen” and “CD152” (see, e.g., Murata (1999) Am. J.Pathol. 155:453-460) are used interchangeably, and include variants,isoforms, species homologs of human CTLA-4, and analogs having at leastone common epitope with CTLA-4 (see, e.g., Balzano (1992) Int. J. CancerSuppl. 7:28-32). A complete sequence of human CTLA-4 is set forth inGenBank Accession No. L1 5006.

The term “antibody” as used to herein may include whole antibodies andany antigen binding fragments (i.e., “antigen-binding portions”) orsingle chains thereof. An “antibody” refers, in one embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Incertain naturally occurring IgG, IgD, and IgA antibodies, the heavychain constant region is comprised of three domains, CH1, CH2 and CH3.In certain naturally occurring antibodies, each light chain is comprisedof a light chain variable region (abbreviated herein as V_(L)) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁷ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less,even more preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸M and 10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens. An antigen is “substantially identical” to a given antigen ifit exhibits a high degree of sequence identity to the given antigen, forexample, if it exhibits at least 80%, at least 90%, preferably at least95%, more preferably at least 97%, or even more preferably at least 99%sequence identity to the sequence of the given antigen. By way ofexample, an antibody that binds specifically to human OX40 maycross-react with OX40 from certain non-human primate species (e.g.,cynomolgus monkey), but may not cross-react with OX40 from other species(e.g., murine OX40), or with an antigen other than OX40.

An immunoglobulin may be from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. The IgGisotype is divided in subclasses in certain species: IgG1, IgG2, IgG3and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. In certainembodiments, the anti-OX40 antibodies described herein are of the IgG1or IgG2 subtype. Immunoglobulins, e.g., IgG1, exist in severalallotypes, which differ from each other in at most a few amino acids.“Antibody” may include, by way of example, both naturally occurring andnon-naturally occurring antibodies; monoclonal and polyclonalantibodies; chimeric and humanized antibodies; human and nonhumanantibodies; wholly synthetic antibodies; and single chain antibodies.

The term “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human OX40). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibody,e.g., an anti-OX40 antibody described herein, include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), CL andCH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These and other potential constructs are described at Chan & Carter(2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtainedusing conventional techniques known to those with skill in the art, andthe fragments are screened for utility in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a particular epitope.Typically such monoclonal antibodies will be derived from a single cellor nucleic acid encoding the antibody, and will be propagated withoutintentionally introducing any sequence alterations. Accordingly, theterm “human monoclonal antibody” refers to a monoclonal antibody thathas variable and optional constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma, for example, obtained by fusing a B cellobtained from a transgenic or transchromosomal non-human animals (e.g.,a transgenic mouse having a genome comprising a human heavy chaintransgene and a light chain), to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutations thatoccur, for example, during antibody maturation. As known in the art(see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid sequences that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not be identical to the original germline sequences,but instead will be substantially identical or similar (i.e., have atleast 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region also is derived from humangermline immunoglobulin sequences. The antibodies described herein mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” antibodies and “fully human” antibodies andare used synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most, or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants within a specificisotype group, which variants differ in a few amino acids (see, e.g.,Jefferis et al. (2009) mAbs 1:1). Antibodies described herein may be ofany allotype. As used herein, antibodies referred to as “IgG1f” or“IgG1.1f” isotype are IgG1 and effectorless IgG1.1 antibodies,respectively, of the allotype “f,” i.e., having 214R, 356E and 358Maccording to the EU index as in Kabat, as shown, e.g., in SEQ ID NO: 5(see underlined residues in SEQ ID NO: 5 of Table 23).

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to OX40 is substantially free of antibodies that specifically bindantigens other than OX40). An isolated antibody that specifically bindsto an epitope of OX40 may, however, have cross-reactivity to other OX40proteins from different species.

As used herein, an antibody that “inhibits binding of OX40-L to OX40” isintended to refer to an antibody that inhibits the binding of OX40-L toOX40, e.g., in binding assays using hOX40-293 cells, with an EC50 ofabout 1 μg/mL or less, such as about 0.9 μg/mL or less, about 0.85 μg/mLor less, about 0.8 μg/mL or less, about 0.75 μg/mL or less, about 0.7μg/mL or less, about 0.65 μg/mL or less, about 0.6 μg/mL or less, about0.55 μg/mL or less, about 0.5 μg/mL or less, about 0.45 μg/mL or less,about 0.4 μg/mL or less, about 0.35 μg/mL or less, about 0.3 μg/mL orless, about 0.25 μg/mL or less, about 0.2 μg/mL or less, about 0.15μg/mL or less, or about 0.1 μg/mL or less, in art-recognized methods,e.g., the FACS-based binding assays described herein.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include C1q binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated phagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIB) receptor. Various properties ofhuman Fc-Rs are summarized in Table 1. The majority of innate effectorcell types coexpress one or more activating FcγR and the inhibitoryFcγRIIB, whereas natural killer (NK) cells selectively express oneactivating Fc receptor (FcγRIII in mice and Fc-RIIIA in humans) but notthe inhibitory FcγRIIB in mice and humans. Human IgG1 binds to mosthuman Fc receptors and is considered equivalent to murine IgG2a withrespect to the types of activating Fc receptors that it binds to.

TABLE 1 Properties of human FcγRs Affinity for Allelic human IsotypeCellular Fcγ variants IgG preference distribution FcγRI None High IgG1 =3 > 4 >> 2 Monocytes, described (K_(D) ~10 macrophages, nM) activatedneutrophils, dentritic cells? FcγRIIA H131 Low to IgG1 > 3 > 2 > 4Neutrophils, medium monocytes, R131 Low IgG1 > 3 > 4 > 2 macrophages,eosinophils, dentritic cells, platelets FcγRIIIA V158 Medium IgG1 = 3 >>4 > 2 NK cells, F158 Low IgG1 = 3 >> 4 > 2 monocytes, macrophages, mastcells, eosinophils, dentritic cells? FcγRIIB 1232 Low IgG1 = 3 = 4 > 2 Bcells, T232 Low IgG1 = 3 = 4 > 2 monocytes, macrophages, dentriticcells, mast cells

An “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc”refers to the C-terminal region of the heavy chain of an antibody thatmediates the binding of the immunoglobulin to host tissues or factors,including binding to Fc receptors located on various cells of the immunesystem (e.g., effector cells) or to the first component (C1q) of theclassical complement system. Thus, an Fc region comprises the constantregion of an antibody excluding the first constant region immunoglobulindomain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fcregion comprises C_(H2) and C_(H3) constant domains in each of theantibody's two heavy chains; IgM and IgE Fc regions comprise three heavychain constant domains (C_(H) domains 2-4) in each polypeptide chain.For IgG, the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 andthe hinge between Cγ1 and Cγ2. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition C226 or P230 (or amino acid between these two amino acids) tothe carboxy-terminus of the heavy chain, wherein the numbering isaccording to the EU index as in Kabat. Kabat et al. (1991) Sequences ofProteins of Immunological Interest, National Institutes of Health,Bethesda, Md.; see also FIGS. 3C-3F of U.S. Pat. App. Pub. No.2008/0248028. The CH2 domain of a human IgG Fc region extends from aboutamino acid 231 to about amino acid 340, whereas the CH3 domain ispositioned on C-terminal side of a C_(H2) domain in an Fc region, i.e.,it extends from about amino acid 341 to about amino acid 447 of an IgG.As used herein, the Fc region may be a native sequence Fc, including anyallotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc).Fc may also refer to this region in isolation or in the context of anFc-comprising protein polypeptide such as a “binding protein comprisingan Fc region,” also referred to as an “Fc fusion protein” (e.g., anantibody or immunoadhesin).

A “hinge”, “hinge domain” or “hinge region” or “antibody hinge region”refers to the domain of a heavy chain constant region that joins the CH1domain to the CH2 domain and includes the upper, middle, and lowerportions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hingeprovides varying levels of flexibility between the binding and effectorregions of an antibody and also provides sites for intermolecular,disulfide bonding between the two heavy chain constant regions. As usedherein, a hinge starts at Glu216 and ends at Gly237 for all IgG isotypes(Roux et al., 1998 J Immunol 161:4083). The sequences of wildtype IgG1,IgG2, IgG3 and IgG4 hinges are shown in Tables 2 and 23.

TABLE 2 Hinge region amino acids C-terminal Ig Type C_(H)1* Upper HingeMiddle Hinge Lower Hinge IgG1 VDKRV EPKSCDKTHT CPPCP APELLGG (SEQ ID NO:(SEQ ID NO: 188) (SEQ ID NO: 192) (SEQ ID NO: 186) 200) IgG2 VDKTV ERKCCVECPPCP APPVAG (SEQ ID NO: (SEQ ID NO: 193) (SEQ ID NO: 187) 201)IgG3 (17-15- VDKRV ELKTPLGDTTHT CPRCP APELLGG 15-15) (SEQ ID NO:(SEQ ID NO: 189) (EPKSCDTPPPCPRCP)₃ (SEQ ID NO: 186) (SEQ ID NO: 194)200) IgG3 (17-15- VDKRV ELKTPLGDTTHT CPRCP APELLGG 15) (SEQ ID NO:(SEQ ID NO: 189) (EPKSCDTPPPCPRCP)₂ (SEQ ID NO: 186) (SEQ ID NO: 195)200) IgG3 (17-15) VDKRV ELKTPLGDTTHT CPRCP APELLGG (SEQ ID NO:(SEQ ID NO: 189) (EPKSCDTPPPCPRCP)₁ (SEQ ID NO: 186) (SEQ ID NO: 196)200) IgG3 (15-15- VDKRV EPKS CDTPPPCPRCP APELLGG 15) (SEQ ID NO:(SEQ ID NO: 190) (EPKSCDTPPPCPRCP)₂ (SEQ ID NO: 186) (SEQ ID NO: 197)200) IgG3 (15) VDKRV EPKS CDTPPPCPRCP APELLGG (SEQ ID NO:(SEQ ID NO: 190) (SEQ ID NO: 198) (SEQ ID NO: 186) 200) IgG4 VDKRVESKYGPP CPSCP APEFLGG (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 199)(SEQ ID NO: 186) 200) *C-terminal amino acid sequences of the CH1domains.

The term “hinge” includes wild-type hinges (such as those set forth inTable 23), as well as variants thereof (e.g., non-naturally-occurringhinges or modified hinges). For example, the term “IgG2 hinge” includeswildtype IgG2 hinge, as shown in Table 23, and variants having 1, 2, 3,4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations, e.g.,substitutions, deletions or additions. Exemplary IgG2 hinge variantsinclude IgG2 hinges in which 1, 2, 3 or all 4 cysteines (C219, C220,C226 and C229) are changed to another amino acid. In a specificembodiment, an IgG2 comprises a C219S substitution. In certainembodiments, a hinge is a hybrid hinge that comprises sequences from atleast two isotypes. For example, a hinge may comprise the upper, middleor lower hinge from one isotype and the remainder of the hinge from oneor more other isotypes. For example, a hinge can be an IgG2/IgG1 hinge,and may comprise, e.g., the upper and middle hinges of IgG2 and thelower hinge of IgG1. A hinge may have effector function or be deprivedof effector function. For example, the lower hinge of wildtype IgG1provides effector function.

The term “CH1 domain” refers to the heavy chain constant region linkingthe variable domain to the hinge in a heavy chain constant domain. Asused herein, a CH1 domain starts at A118 and ends at V215. The term “CH1domain” includes wildtype CH1 domains (such as having SEQ ID NO: 202 forIgG1 and SEQ ID NO: 203 for IgG2; Table 23), as well as variants thereof(e.g., non-naturally occurring CH1 domains or modified CH1 domains). Forexample, the term “CH1 domain” includes wildtype CH1 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH1 domains include CH1 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. Modificationsto the CH1 domain that affect a biological activity of an antibody areprovided herein.

The term “CH2 domain” refers to the heavy chain constant region linkingthe hinge to the CH3 domain in a heavy chain constant domain. As usedherein, a CH2 domain starts at P238 and ends at K340. The term “CH2domain” includes wildtype CH2 domains (such as having SEQ ID NO: 204 forIgG1 and SEQ ID NO: 205 for IgG2; Table 23), as well as variants thereof(e.g., non-naturally occurring CH2 domains or modified CH2 domains). Forexample, the term “CH2 domain” includes wildtype CH2 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH2 domains include CH2 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. In certainembodiments, a CH2 domain comprises the substitutions A330S/P331S thatreduce effector function. Other modifications to the CH2 domain thataffect a biological activity of an antibody are provided herein.

The term “CH3 domain” refers to the heavy chain constant region that isC-terminal to the CH2 domain in a heavy chain constant domain. As usedherein, a CH3 domain starts at G341 and ends at K447. The term “CH3domain” includes wildtype CH3 domains (such as having SEQ ID NO: 206 forIgG1 and SEQ ID NO: 207 for IgG2; Table 23), as well as variants thereof(e.g., non-naturally occurring CH3 domains or modified CH3 domains). Forexample, the term “CH3 domain” includes wildtype CH3 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH3 domains include CH3 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. Modificationsto the CH3 domain that affect a biological activity of an antibody areprovided herein.

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fcs include the various allotypes of Fcs (see, e.g., Jefferiset al. (2009) mAbs 1:1).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., OX40) to which an immunoglobulin or antibody specificallybinds. Epitopes within protein antigens can be formed both fromcontiguous amino acids (usually a linear epitope) or noncontiguous aminoacids juxtaposed by tertiary folding of the protein (usually aconformational epitope). Epitopes formed from contiguous amino acids aretypically, but not always, retained on exposure to denaturing solvents,whereas epitopes formed by tertiary folding are typically lost ontreatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods for determining what epitopes arebound by a given antibody (i.e., epitope mapping) are well known in theart and include, for example, immunoblotting and immunoprecipitationassays, wherein overlapping or contiguous peptides (e.g., from OX40) aretested for reactivity with a given antibody (e.g., an anti-OX40antibody). Methods of determining spatial conformation of epitopesinclude techniques in the art and those described herein, for example,x-ray crystallography, 2-dimensional nuclear magnetic resonance andHDX-MS (see, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants on the antigen involved in antibody-antigenrecognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same group of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on OX40” withthe antibodies described herein include art-recognized epitope mappingmethods, such as x-ray analyses of crystals of antigen:antibodycomplexes which provides atomic resolution of the epitope andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsmonitor the binding of the antibody to antigen fragments (e.g.,proteolytic fragments) or to mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue withinthe antigen sequence is often considered an indication of an epitopecomponent (e.g., alanine scanning mutagenesis—Cunningham & Wells (1985)Science 244:1081). In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest to affinity isolate specific short peptidesfrom combinatorial phage display peptide libraries. Antibodies havingthe same or closely related VH and VL or the same CDR1, 2 and 3sequences are expected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that (partially or completely) inhibit the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known competition experiments. In certain embodiments,an antibody competes with, and inhibits binding of another antibody to atarget by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.The level of inhibition or competition may be different depending onwhich antibody is the “blocking antibody” (i.e., the cold antibody thatis incubated first with the target). Competition assays can be conductedas described, for example, in Ed Harlow and David Lane, Cold Spring HarbProtoc; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “UsingAntibodies” by Ed Harlow and David Lane, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA 1999. Competing antibodies bind tothe same epitope, an overlapping epitope, or to adjacent epitopes (e.g.,as evidenced by steric hindrance).

Other art-recognized competitive binding assays include: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli et al., Methods in Enzymology 9:242 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986));solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press (1988)); solid phase direct label RIA using I-125 label(see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directlabeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen but not to other antigens.Typically, the antibody (i) binds with an equilibrium dissociationconstant (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by, e.g., surface plasmon resonance (SPR) technology in aBIACORE 2000 surface plasmon resonance (SPR) instrument using thepredetermined antigen, e.g., recombinant human OX40, as the analyte andthe antibody as the ligand, and (ii) binds to the predetermined antigenwith an affinity that is at least two-fold greater than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. Accordingly, anantibody that “specifically binds to human OX40” refers to an antibodythat binds to soluble or cell bound human OX40 with a K_(D) of 10⁻⁷ M orless, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or evenlower. An antibody that “cross-reacts with cynomolgus OX40” refers to anantibody that binds to cynomolgus OX40 with a K_(D) of 10⁻⁷ M or less,such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.In certain embodiments, antibodies that do not cross-react with OX40from a non-human species (e.g., murine OX40) exhibit essentiallyundetectable binding against these proteins in standard binding assays.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate constant of a particular antibody-antigeninteraction, whereas the term “k_(dis)” or “k_(d),” as used herein, isintended to refer to the dissociation rate constant of a particularantibody-antigen interaction. The term “K_(D)”, as used herein, isintended to refer to the equilibrium dissociation constant, which isobtained from the ratio of k_(d) to k_(a) (i.e., k_(d)/k_(a)) and isexpressed as a molar concentration (M). K_(D) values for antibodies canbe determined using methods well established in the art. A preferredmethod for determining the K_(D) of an antibody is by using surfaceplasmon resonance, preferably using a biosensor system such as aBiacore® SPR system or flow cytometry and Scatchard analysis.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M orless.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof, refers to theconcentration of an antibody or an antigen-binding portion thereof thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

The term “binds to immobilized OX40,” refers to the ability of anantibody described herein to bind to OX40, for example, expressed on thesurface of a cell or attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody described herein to bind to OX40 from a different species. Forexample, an antibody described herein that binds human OX40 may alsobind OX40 from another species (e.g., cynomolgus OX40). As used herein,cross-reactivity may be measured by detecting a specific reactivity withpurified antigen in binding assays (e.g., SPR, ELISA) or binding to, orotherwise functionally interacting with, cells physiologicallyexpressing OX40. Methods for determining cross-reactivity includestandard binding assays as described herein, for example, by BIACORE®surface plasmon resonance (SPR) analysis using a BIACORE® 2000 SPRinstrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein may contain amodification such as, but not limited to, glycosylation, phosphorylationor a disulfide bond. A “protein” may comprise one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, and may be cDNA.

Also provided are “conservative sequence modifications” of the sequencesset forth herein, e.g., in Table 23, i.e., nucleotide and amino acidsequence modifications which do not abrogate the binding of the antibodyencoded by the nucleotide sequence or containing the amino acidsequence, to the antigen. Such conservative sequence modificationsinclude conservative nucleotide and amino acid substitutions, as wellas, nucleotide and amino acid additions and deletions. For example,modifications can be introduced into a sequence in Table 23 by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative sequence modifications includeconservative amino acid substitutions, in which the amino acid residueis replaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an anti-OX40 antibody ispreferably replaced with another amino acid residue from the same sidechain family. Methods of identifying nucleotide and amino acidconservative substitutions that do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Alternatively, inanother embodiment, mutations can be introduced randomly along all orpart of an anti-OX40 antibody coding sequence, such as by saturationmutagenesis, and the resulting modified anti-OX40 antibodies can bescreened for improved binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For polypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of theamino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences when the sequences areoptimally aligned (i.e., % homology=# of identical positions/total # ofpositions×100), with optimal alignment determined taking into accountthe number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, e.g., cDNA, may be mutated, in accordance with standardtechniques to provide gene sequences. For coding sequences, thesemutations, may affect amino acid sequence as desired. In particular, DNAsequences substantially homologous to or derived from native V, D, J,constant, switches and other such sequences described herein arecontemplated.

The term “vector” as used herein, is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and maybe a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. An antigenmay be OX40 or a fragment thereof. An antigen may also be a tumorantigen, against which protective or therapeutic immune responses aredesired, e.g., antigens expressed by a tumor cell (e.g., for use as atumor vaccine to be administered in combination with an anti-OX40antibody). Antigens include tumor-associated antigens for the preventionor treatment of cancers. Examples of tumor-associated antigens include,but are not limited to, sequences comprising all or part of thesequences of βhCG, gp100 or Pmel17, HER2/neu, WT1, mesothelin, CEA,gp100, MART1, TRP-2, melan-A, NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250),idiotype, MAGE-1, MAGE-3, MAGE-A3, Tyrosinase, Telomerase, SSX2 andMUC-1 antigens, and germ cell derived tumor antigens. Tumor associatedantigens also include the blood group antigens, for example, Le^(a),Le^(b) LeX, LeY, H-2, B-1, B-2 antigens. Alternatively, more than oneantigen can be included in a construct. For example, a MAGE antigen canbe combined with other antigens such as melanin A, tyrosinase, and gp100along with adjuvants such as GM-CSF or IL-12, and linked to an anti-APCantibody.

Sequences of the foregoing antigens are well known in the art. Forexample, an example of a MAGE-3 cDNA sequence is provided in U.S. Pat.No. 6,235,525 (Ludwig Institute for Cancer Research); examples ofNY-ESO-1 nucleic acid and protein sequences are provided in U.S. Pat.Nos. 5,804,381 and 6,069,233 (Ludwig Institute for Cancer Research);examples of Melan-A nucleic acid and protein sequences are provided inU.S. Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Institute for CancerResearch); examples of NY-BR-1 nucleic acid and protein sequences areprovided in U.S. Pat. Nos. 6,774,226 and 6,911,529 (Ludwig Institute forCancer Research) and examples of NY-CO-58 nucleic acid and proteinsequences are provided in WO 02/90986 (Ludwig Institute for CancerResearch); an example of an amino acid sequence for the HER-2/neuprotein is available at GENBANK® Accession No. AAA58637; and anucleotide sequence (mRNA) for human carcinoembryonic antigen-like 1(CEA-1) is available at GENBANK® Accession No. NM020219.

An “immune response” refers to a biological response within a vertebrateagainst foreign agents, which response protects the organism againstthese agents and diseases caused by them. An immune response is mediatedby the action of a cell of the immune system (for example, a Tlymphocyte, B lymphocyte, natural killer (NK) cell, macrophage,eosinophil, mast cell, dendritic cell or neutrophil) and solublemacromolecules produced by any of these cells or the liver (includingantibodies, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom the vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues. An immune reaction includes, e.g., activation or inhibition ofa T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+T cell, or the inhibition of a Treg cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., acomponent of a signaling pathway, that may be involved in modulating,regulating, or modifying an immune response. “Modulating,” “regulating,”or “modifying” an immune response refers to any alteration in a cell ofthe immune system or in the activity of such cell (e.g., an effector Tcell). Such modulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunomodulators have been identified,some of which may have enhanced function in a tumor microenvironment. Inpreferred embodiments, the immunomodulator is located on the surface ofa T cell. An “immunomodulatory target” or “immunoregulatory target” isan immunomodulator that is targeted for binding by, and whose activityis altered by the binding of, a substance, agent, moiety, compound ormolecule. Immunomodulatory targets include, for example, receptors onthe surface of a cell (“immunomodulatory receptors”) and receptorligands (“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) with cytolytic activities as well as T helper (Th) cells, whichsecrete cytokines and activate and direct other immune cells, but doesnot include regulatory T cells (Treg cells). Anti-OX40 antibodiesdescribed herein activate T_(eff) cells, e.g., CD4+ and CD8+ T_(eff)cells.

An increased ability to stimulate an immune response or the immunesystem, can result from an enhanced agonist activity of T cellcostimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability to stimulate an immuneresponse or the immune system may be reflected by a fold increase of theEC₅₀ or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD107a or granzymes) and proliferation. Theability to stimulate an immune response or the immune system activitymay be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 foldor more.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8⁺ cells) andhelper T cells (e.g., CD4⁺ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of OX40-L to OX40 on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. In certain embodiments, the anti-OX40 antibodyinhibits binding of OX40-L to OX40 by at least about 50%, for example,about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, determined, e.g., asfurther described herein. In certain embodiments, the anti-OX40 antibodyinhibits binding of OX40-L to OX40 by no more than 50%, for example, byabout 40%, 30%, 20%, 10%, 5% or 1%, determined, e.g., as furtherdescribed herein.

As used herein, the term “inhibits growth” of a tumor includes anymeasurable decrease in the growth of a tumor, e.g., the inhibition ofgrowth of a tumor by at least about 10%, for example, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 99%, or 100%.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division may result in the formation of malignant tumors or cellsthat invade neighboring tissues and may metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with adisease. Prophylaxis refers to administration to a subject who does nothave a disease, to prevent the disease from occurring or minimize itseffects if it does.

A “hematological malignancy” includes a lymphoma, leukemia, myeloma or alymphoid malignancy, as well as a cancer of the spleen and the lymphnodes. Exemplary lymphomas include both B cell lymphomas (a B-cellhematological cancer) and T cell lymphomas. B-cell lymphomas includeboth Hodgkin's lymphomas and most non-Hodgkin's lymphomas. Non-limitingexamples of B cell lymphomas include diffuse large B-cell lymphoma,follicular lymphoma, mucosa-associated lymphatic tissue lymphoma, smallcell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia),mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B celllymphoma, Waldenström macroglobulinemia, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis.Non-limiting examples of T cell lymphomas include extranodal T celllymphoma, cutaneous T cell lymphomas, anaplastic large cell lymphoma,and angioimmunoblastic T cell lymphoma. Hematological malignancies alsoinclude leukemia, such as, but not limited to, secondary leukemia,chronic lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, and acute lymphoblastic leukemia. Hematologicalmalignancies further include myelomas, such as, but not limited to,multiple myeloma and smoldering multiple myeloma. Other hematologicaland/or B cell- or T-cell-associated cancers are encompassed by the termhematological malignancy.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.

In reference to solid tumors, an effective amount comprises an amountsufficient to cause a tumor to shrink and/or to decrease the growth rateof the tumor (such as to suppress tumor growth) or to prevent or delayother unwanted cell proliferation. In certain embodiments, an effectiveamount is an amount sufficient to delay tumor development. In someembodiments, an effective amount is an amount sufficient to prevent ordelay tumor recurrence. An effective amount can be administered in oneor more administrations. The effective amount of the drug or compositionmay: (i) reduce the number of cancer cells; (ii) reduce tumor size;(iii) inhibit, retard, slow to some extent and may stop cancer cellinfiltration into peripheral organs; (iv) inhibit, i.e., slow to someextent and may stop, tumor metastasis; (v) inhibit tumor growth; (vi)prevent or delay occurrence and/or recurrence of tumor; and/or (vii)relieve to some extent one or more of the symptoms associated with thecancer. In one example, an “effective amount” is the amount of anti-OX40antibody and the amount of anti-PD-1 antibody (e.g., nivolumab) oranti-CTLA-4 antibody (e.g., ipilimumab), in combination, clinicallyproven to affect a significant decrease in cancer or slowing ofprogression of cancer, such as an advanced solid tumor. As used herein,the terms “fixed dose”, “flat dose” and “flat-fixed dose” are usedinterchangeably and refer to a dose that is administered to a patientwithout regard for the weight or body surface area (BSA) of the patient.The fixed or flat dose is therefore not provided as a mg/kg dose, butrather as an absolute amount of the agent.

As used herein, a “body surface area (BSA)-based dose” refers to a dosethat is adjusted to the body-surface area (BSA) of the individualpatient. A BSA-based dose may be provided as mg/kg body weight. Variouscalculations have been published to arrive at the BSA without directmeasurement, the most widely used of which is the Du Bois formula (seeDu Bois D, Du Bois E F (June 1916) Archives of Internal Medicine 17 (6):863-71; and Verbraecken, J. et al. (April 2006). Metabolism—Clinical andExperimental 55 (4): 515-24). Other exemplary BSA formulas include theMosteller formula (Mosteller R D. N Engl J Med., 1987; 317:1098), theHaycock formula (Haycock G B, et al., J Pediatr 1978, 93:62-66), theGehan and George formula (Gehan E A, George S L, Cancer Chemother Rep1970, 54:225-235), the Boyd formula (Current, J D (1998), The InternetJournal of Anesthesiology 2 (2); and Boyd, Edith (1935), University ofMinnesota. The Institute of Child Welfare, Monograph Series, No. x.London: Oxford University Press), the Fujimoto formula (Fujimoto S, etal., Nippon Eiseigaku Zasshi 1968; 5:443-50), the Takahira formula(Fujimoto S, et al., Nippon Eiseigaku Zasshi 1968; 5:443-50), and theSchlich formula (Schlich E, et al., Ernährungs Umschau 2010;57:178-183).

A “prophylactically effective amount” or a “prophylactically effectivedosage” of a drug, is an amount of the drug that, when administeredalone or in combination with another therapeutic agent to a subject atrisk of developing a disease or of suffering a recurrence of disease,inhibits the development or recurrence of the disease. The ability of atherapeutic or prophylactic agent to promote disease regression orinhibit the development or recurrence of the disease can be evaluatedusing a variety of methods known to the skilled practitioner, such as inhuman subjects during clinical trials, in animal model systemspredictive of efficacy in humans, or by assaying the activity of theagent in in vitro assays.

By way of example, an anti-cancer agent is a drug that slows cancerprogression or promotes cancer regression in a subject. In preferredembodiments, a therapeutically effective amount of the drug promotescancer regression to the point of eliminating the cancer. “Promotingcancer regression” means that administering an effective amount of thedrug, alone or in combination with an anti-neoplastic agent, results ina reduction in tumor growth or size, necrosis of the tumor, a decreasein severity of at least one disease symptom, an increase in frequencyand duration of disease symptom-free periods, a prevention of impairmentor disability due to the disease affliction, or otherwise ameliorationof disease symptoms in the patient. Pharmacological effectiveness refersto the ability of the drug to promote cancer regression in the patient.Physiological safety refers to an acceptably low level of toxicity, orother adverse physiological effects at the cellular, organ and/ororganism level (adverse effects) resulting from administration of thedrug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug preferably inhibits cell growthor tumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. In themost preferred embodiments, a therapeutically effective amount or dosageof the drug completely inhibits cell growth or tumor growth, i.e.,preferably inhibits cell growth or tumor growth by 100%. The ability ofa compound to inhibit tumor growth can be evaluated using the assaysdescribed infra. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. In other preferred embodiments described herein,tumor regression may be observed and may continue for a period of atleast about 20 days, more preferably at least about 40 days, or evenmore preferably at least about 60 days.

The terms “patient” and “subject” refer to any human or non-human animalthat receives either prophylactic or therapeutic treatment. For example,the methods and compositions described herein can be used to treat asubject or patient having cancer, such as an advanced solid tumor. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-OX40 Antibodies

Described herein are antibodies, e.g., fully human antibodies, which arecharacterized by particular functional features or properties. Forexample, the antibodies specifically bind human OX40. Additionally,antibodies may cross react with OX40 from one or more non-humanprimates, such as cynomolgus OX40. Such antibodies are useful in thetreatment of cancer when used as monotherapy, or when used incombination with an immuno-oncology agent, such as an anti-PD-1 antibody(e.g., nivolumab) or anti-CTLA-4 antibody (e.g., ipilimumab).

Anti-OX40 antibodies described herein exhibit one or more or all of thefollowing functional properties:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by BIACORE®        SPR analysis;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

Preferably, the antibodies bind to OX40 with high affinity, for example,with a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ Mor less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, or 10⁻⁹ M to 10⁻⁷ M. In certain embodiments,an anti-OX40 antibody binds to soluble human OX40, e.g., as determinedby BIACORE® SPR analysis, with a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M orless, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ Mto 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, or 10⁻⁸ M to 10⁻⁷ M. Incertain embodiments, the anti-OX40 antibody binds to bound (e.g., cellmembrane bound) human OX40, such as on activated human T cells, e.g., asdetermined by flow cytometry, with a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M orless, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ Mto 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸ M, 10⁻¹¹ M to 10⁻⁹ M, or10⁻¹⁰ M to 10⁻⁹ M. In certain embodiments, an anti-OX40 antibody bindsto bound (e.g., cell membrane bound) human OX40, such as on activatedhuman T cells, e.g., as determined by FACS, with an EC₅₀ of 10⁻⁷ M orless, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸ M, 10⁻¹¹ Mto 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M. In certain embodiments, the anti-OX40antibody binds to soluble human OX40 with a K_(D) of 10⁻⁷ M or less,10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, or 10⁻⁸ M to10⁻⁷ M, and to cell membrane bound human OX40 with a K_(D) or EC50 of10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less,10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸M, 10⁻¹¹ M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M.

Anti-OX40 antibodies described herein may bind to cynomolgus OX40, e.g.,bind to membrane bound cynomolgus OX40, e.g., with an EC₅₀ of 100 nM orless, 10 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1nM, or 10 nM to 1 nM, e.g., as measured by FACS (e.g., as described inthe Examples).

Anti-OX40 antibodies described herein may stimulate or enhance an immuneresponse, e.g., by activating T_(eff) cells, limiting the suppression ofTeffector cells by Treg cells, depleting and/or inhibiting tumor Tregcells and/or activating NK cells, e.g., in the tumor. For example, theanti-OX40 antibodies may activate or costimulate T_(eff) cells asevidenced, e.g., by enhanced cytokine (e.g., IL-2 and IFN-γ) secretionand/or enhanced proliferation. In certain embodiments, CD3 stimulationis also provided. In certain embodiments, the OX40 antibody increasesIL-2 secretion by a factor of 50%, 100% (i.e., 2 fold), 3 fold, 4 fold,5 fold or more, optionally with a maximum of up to 10 fold, 30 fold, 100fold, as measured, e.g., on primary human T cells or T cells expressinghuman OX40 (e.g., as further described in the Examples). In certainembodiments, the OX40 antibody increases IFN-γ secretion by a factor of50%, 100% (i.e., 2 fold), 3 fold, 4 fold, 5 fold or more, optionallywith a maximum of up to 10 fold, 30 fold, 100 fold, as measured, e.g.,on primary human T cells or T cells expressing human OX40 (e.g., asfurther described in the Examples).

Anti-OX40 antibodies described herein may inhibit binding of human OX40Lto human OX40 on cells, e.g., 293 cells expressing human OX40 (i.e.,hOX40-293 cells), e.g., with an EC₅₀ of 10 nM or less, 1 nM or less,0.01 nM to 10 nM, 0.1 nM to 10 nM, or 0.1 nM to 1 nM (see Example 6).

Anti-OX40 antibodies described herein bind to an epitope on OX40, asdetermined, for example, by binding to fragments of human OX40. Forexample, in certain embodiments, the antibody binds to all or a portionof the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ IDNO: 2) as determined, for example, by HDX or by binding of theantibodies to fragments of human OX40, followed by enzymatic cleavage(see Example 11). In other embodiments, the antibody binds to all or aportion of the sequence DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO:179) of human OX40 (SEQ ID NO: 2).

In certain embodiments, the anti-OX40 antibodies described herein bindto all or a portion of the sequence SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR(SEQ ID NO: 182).

In other embodiments, the anti-OX40 antibodies described herein bind toall or a portion of the sequence PCKPCTWCNLR (SEQ ID NO: 183).

In yet other embodiments, the anti-OX40 antibodies that bind to all or aportion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) further bindto all or a portion of the sequence QLCTATQDTVCR (SEQ ID NO: 184).

In additional embodiments, the anti-OX40 antibodies described hereinbind to all or a portion of the sequence SQNTVCRPCGPGFYN (SEQ ID NO:185).

Anti-OX40 antibodies described herein may compete for binding to OX40with (or inhibit binding of) anti-OX40 antibodies comprising CDRs orvariable regions described herein, e.g., 3F4, 14B6-1, 14B6-2, 23H3,6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1. In certainembodiments, anti-OX40 antibodies inhibit binding of 3F4, 14B6-1,14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or20C1 to human OX40 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or by 100%. In certain embodiments, 3F4, 14B6-1, 14B6-2, 23H3,6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 inhibit bindingof anti-OX40 antibodies to human OX40 by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or by 100%.

In certain embodiments, the antibodies induce or enhance T cellactivation with multivalent crosslinking through, e.g., FcR binding. Incertain embodiments, the antibodies are multivalent, e.g., bivalent. Incertain embodiments, the antibodies are not monovalent.

In certain embodiments, the antibodies have 1, 2, 3, 4, 5, or 6 of thefollowing features:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by BIACORE®        SPR analysis;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

Accordingly, an antibody that exhibits one or more of these functionalproperties (e.g., biochemical, immunochemical, cellular, physiologicalor other biological activities, or the like) as determined according tomethodologies known to the art and described herein, will be understoodto relate to a statistically significant difference in the particularactivity relative to that seen in the absence of the antibody (e.g., orwhen a control antibody of irrelevant specificity is present).Preferably, the anti-OX40 antibody increases a measured parameter (e.g.,T cell proliferation, cytokine production) by at least 10% of themeasured parameter, more preferably by at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 100% (i.e, 2 fold), 3 fold, 5 fold, or 10 fold.Conversely, the antibody may decrease a measured parameter (e.g., tumorvolume, OX40-L binding to OX40, quantity of regulatory T cells intumors) by at least 10% of the measured parameter, more preferably by atleast 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, 95%, or 99%.

Standard assays to evaluate the binding ability of the antibodies towardOX40 of various species are known in the art, including for example,ELISAs, Western blots, and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by BIACORE® SPR analysis. Assays to evaluate the effects of theantibodies on functional properties of OX40 (e.g., ligand binding, Tcell proliferation, cytokine production) are described in further detailinfra and in the Examples.

In certain embodiments, the anti-OX40 antibodies are not nativeantibodies or are not naturally-occurring antibodies, e.g., anti-OX40antibodies with post-translational modifications that are different fromthose of antibodies that are naturally occurring, such as by havingmore, less, or a different type of post-translational modification.

II. Exemplary Anti-OX40 Antibodies

Particular antibodies described herein are antibodies, e.g., monoclonalantibodies, having the CDR and/or variable region sequences ofantibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,14A2-1, 14A2-2, and 20C1, isolated and structurally characterized asdescribed in Example 1, as well as antibodies having at least 80%identity (e.g., at least 85%, at least 90%, at least 95%, or at least99% identity) to the variable region or CDR sequences of antibodies 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1. The V_(H) amino acid sequences of 3F4, 14B6 (14B6-1 and14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and14A2-2), and 20C1 are set forth in SEQ ID NOs: 17, 28, 37, 48, 57, 65,73, 84, and 93, respectively. The V_(L) amino acid sequences of 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1 are set forth in SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66,74, 85, 86, and 94, respectively.

Accordingly, provided herein are antibodies, or antigen binding portionthereof, comprising heavy and light chain variable regions, wherein theheavy chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 17, 28, 37, 48, 57, 65, 73, 84,and 93.

Also provided are antibodies, or antigen binding portions thereof,comprising heavy and light chain variable regions, wherein the lightchain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66, 74, 85,86, and 94.

Provided herein are antibodies, or antigen-binding portion thereof,comprising: heavy and light chain variable region sequences comprisingSEQ ID NOs: 17 and 18; 28 and 29; 28 and 30; 37 and 38; 48 and 49; 48and 50; 57 and 58; 65 and 66; 73 and 74; 84 and 85; 84 and 86; 93 and94.

Anti-OX40 antibodies described herein may comprise the heavy and lightchain CDR1s, CDR2s and CDR3s of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, or combinations thereof. Theamino acid sequences of the V_(H) CDR1s of 3F4, 14B6 (14B6-1 and14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and14A2-2), and 20C1 are set forth in SEQ ID NOs: 11, 19, 31, 39, 51, 59,67, 75, and 87, respectively. The amino acid sequences of the V_(H)CDR2s of 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2),18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2), and 20C1 are set forth inSEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, and 88, respectively. Theamino acid sequences of the V_(H) CDR3s of 3F4, 14B6 (14B6-1 and14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and14A2-2), and 20C1 are set forth in SEQ ID NOs: 13, 21, 33, 41, 53, 61,69, 77, and 89. The amino acid sequences of the V_(L) CDR1s of 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1 are set forth in SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62,70, 78, 81, and 90, respectively. The amino acid sequences of the V_(L)CDR2s of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,14A2-1, 14A2-2, and 20C1 are set forth in SEQ ID NOs: 15, 23, 26, 35,43, 46, 55, 63, 71, 79, 82, and 91, respectively. The amino acidsequences of the V_(L) CDR3s of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 are set forth in SEQ ID NOs:16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92, respectively. TheCDR regions are delineated using the Kabat system (Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242).

Given that each of these antibodies bind to OX40 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3) to create other anti-OX40binding antibodies. OX40 binding of such “mixed and matched” antibodiescan be tested using the binding assays described above and in theExamples (e.g., ELISAs). Preferably, when V_(H) CDR sequences are mixedand matched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(L) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(L) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 3F4, 14B6-1,14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1.“Mixed and matched” antibodies having binding affinity, bioactivityand/or other properties equivalent or superior to the specificantibodies disclosed herein may be selected for use in the methods ofthe present invention.

Provided herein are isolated antibodies, or antigen binding portionthereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 19, 31, 39, 51,59, 67, 75, and 87;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 12, 20, 32, 40, 52,60, 68, 76, and 88;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 21, 33, 41, 53,61, 69, 77, and 89;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 14, 22, 25, 34, 42,45, 54, 62, 70, 78, 81, and 90;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 15, 23, 26, 35, 43,46, 55, 63, 71, 79, 82, and 91; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 24, 27, 36, 44,47, 56, 64, 72, 80, 83, and 92;

wherein the antibody specifically binds to human OX40.

In one embodiment, the antibody comprises heavy and light chain variableregions, wherein the heavy chain variable region CDR1, CDR2, and CDR3regions comprise: SEQ ID NOs: 11-13; 19-21; 31-33; 39-41; 51-53; 59-61;67-69; 74-77; 87-89; and 87, 317, and 89, respectively;

wherein the antibody specifically binds to human OX40.

In another embodiment, the antibody comprises heavy and light chainvariable regions, wherein the light chain variable region CDR1, CDR2,and CDR3 regions comprise: SEQ ID NOs: 14-16; 22-24; 25-27; 34-36;42-44; 45-47; 54-56; 62-64; 70-72; 78-80; 81-83; and 90-92,respectively;

wherein the antibody specifically binds to human OX40.

In a particular embodiment, the antibody comprises heavy and light chainvariable regions, wherein the antibody comprises:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87, 317, and 89, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:11-13, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 14-16, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 22-24, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 25-27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:31-33, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 34-36, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 42-44, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 45-47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:51-53, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 54-56, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:59-61, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 62-64, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:67-69, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 70-72, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 78-80, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 81-83, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87-89, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 90-92, respectively;

wherein the antibody specifically binds to human OX40.

In another embodiment, the antibody comprises heavy and light chainvariable regions, wherein the antibody comprises:

(a) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:87, 317, and 89, respectively, and/or light chain CDR1, CDR2, and CDR3sequences consisting of SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:11-13, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 14-16, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 22-24, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 25-27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:31-33, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 34-36, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 42-44, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 45-47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:51-53, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 54-56, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:59-61, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 62-64, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:67-69, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 70-72, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 78-80, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 81-83, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:87-89, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 90-92, respectively.

A VH domain, or one or more CDRs thereof, described herein may be linkedto a constant domain for forming a heavy chain, e.g., a full lengthheavy chain. Similarly, a VL domain, or one or more CDRs thereof,described herein may be linked to a constant domain for forming a lightchain, e.g., a full length light chain. A full length heavy chain (withthe exception of the C-terminal lysine (K) or with the exception of theC-terminal glycine and lysine (GK), which may be absent) and full lengthlight chain combine to form a full length antibody. N-terminal glutamineand glutamate residues may also be converted to pyroglutamate residueson both light and heavy chains.

A VH domain described herein may be fused to the constant domain of ahuman IgG, e.g., IgG1, IgG2, IgG3 or IgG4, which are eithernaturally-occurring or modified, e.g., as further described herein. Forexample, a heavy chain may comprise the amino acid sequence of any VHdomain described herein fused to the human IgG1 amino acid sequence setforth in SEQ ID NO: 5.

The human IgG1 constant domain may also be that of an allotypic variant.For example, an allotypic variant of IgG1 comprises an R107K, E189D andM191L (underlined above, with numbering according to that in SEQ ID NO:6). Within the full length heavy region, these amino acid substitutionsare numbered R214K, E356D and M358L.

A VL domain described herein may be fused to the constant domain of ahuman kappa or lambda light chain. For example, a light chain maycomprise the amino acid sequence of any VL domain described herein fusedto the human IgG1 kappa light chain amino acid sequence set forth in SEQID NO: 7.

In certain embodiments, the heavy chain constant region comprises alysine or another amino acid at the C-terminus, e.g., it comprises thefollowing last amino acids: LSPGK (SEQ ID NO: 8) for the heavy chain. Incertain embodiments, the heavy chain constant region is lacking one ormore amino acids at the C-terminus, and has, e.g., the C-terminalsequence LSPG (SEQ ID NO: 9) or LSP.

The amino acid sequences of exemplary heavy and light chains are setforth in Table 23 and correspond to SEQ ID NOs: 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125 for theheavy chains and SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, and 122 for the light chains.

Heavy and light chains comprising an amino acid sequence that is atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical toany of the heavy or light chains set forth in Table 23 (or theirvariable regions), e.g., SEQ ID NOs: 95 and 96; 97 and 98; 99 and 100;101 and 102; 103 and 104; 105 and 106; 107 and 108; 109 and 110; 111 and112; 113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and 122;123 and 116; 124 and 116; and 125 and 116 may be used for forminganti-human OX40 antibodies having the desired characteristics, e.g.,those further described herein. Exemplary variants are those comprisingan allotypic variation, e.g., in the constant domain, and/or a mutationin the variable or constant regions, such as the mutations disclosedherein. Heavy and light chains comprising an amino acid sequence thatdiffers in at most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1amino acid (by substitution, addition or deletion) from any of the heavyor light chains set forth in Table 23 (or their variable regions) may beused for forming anti-human OX40 antibodies having the desiredcharacteristics, e.g., those further described herein.

In various embodiments, the antibodies described above exhibit one ormore, two or more, three or more, four or more, five or more, six, orall of the following functional properties:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

Such antibodies include, for example, human antibodies, humanizedantibodies, or chimeric antibodies.

In certain embodiments, the anti-OX40 antibodies described herein bindto amino acid residues within the following region of mature human OX40(SEQ ID NO: 2):

(SEQ ID NO: 178) DVVSSKPCKPCTWCNLR,corresponding to amino acid residues 46-62 of mature human OX40 (SEQ IDNO: 2).

In certain embodiments, the anti-OX40 antibodies described herein bindto amino acid residues within the following region of mature human OX40(SEQ ID NO: 2):

(SEQ ID NO: 179) DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK,corresponding to amino acid residues 89-124 of mature human OX40 (SEQ IDNO: 2).

In certain embodiments, the anti-OX40 antibodies described herein thatbind to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:178) bind to all or a portion of the sequenceSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182).

In other embodiments, the anti-OX40 antibodies described herein thatbind to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:178) bind to all or a portion of the sequence PCKPCTWCNLR (SEQ ID NO:183).

In yet other embodiments, the anti-OX40 antibodies that bind to all or aportion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) further bindto all or a portion of the sequence QLCTATQDTVCR (SEQ ID NO: 184).

In additional embodiments, the anti-OX40 antibodies described hereinthat bind to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ IDNO: 178) further bind to all or a portion of the sequenceSQNTVCRPCGPGFYN (SEQ ID NO: 185).

Modified Heavy Chain Constant Domains

The heavy chain constant region of anti-OX40 antibodies described hereinmay be of any isotype, e.g., IgG1, IgG2, IgG3 and IgG4, or combinationsthereof and/or modifications thereof. In certain embodiments, anti-OX40antibodies comprise a modified heavy chain constant region that altersthe properties of the antibody.

As discussed further herein and in the Examples, cross-linking ofanti-OX40 antibodies with unmodified hIgG1 constant regions (hIgG1isotype antibodies) induce OX40 signaling and promote T cell activation,and specifically to promote T cell proliferation, IFN-γ secretion, andIL-2 secretion. Cross-linking can occur by, e.g., binding to human CD32AFcγ receptors (FcγRs) expressed on the surface of transfected CHO cellsin an assay utilizing co-cultures of CHO-CD3-CD32A cells and humanprimary CD4 T cells. Cross-linking can also occur, e.g., by adding asoluble polyclonal anti-human Fcγ antibody in cultures of staphyloccusenterotoxin B (SEB)-activated human peripheral blood mononuclear cells(PBMCs).

Anti-OX40 antibodies with modified heavy chain constant regions (e.g.,IgG1 constant region wherein the CH1/hinge region is replaced with anhIgG2 CH1/hinge region) may have the ability to alter the activities ofthe antibodies relative to antibodies with a fully IgG1 heavy chainconstant region. Exemplary activities that may be altered include, butare not limited to, (1) T cell activation in the presence or absence ofcross-linking, (2) T cell proliferation in the presence or absence ofcross-linking, and/or (3) cytokine secretion (e.g., IFN-γ, IL-2) in thepresence or absence of cross-linking. The methods described in theExamples can be used to determine whether the anti-OX40 antibodies withmodified heavy chain constant regions exhibit these altered activities(see, e.g., Example 27). In preferred embodiments, these alteredactivities do not markedly affect the antigen-binding properties of theantibodies, which can be assessed using, e.g., FACS, SPR).

Accordingly, provided herein are methods of altering the activity ofanti-OX40 antibodies comprising providing an anti-OX40 antibody that hasa non-IgG2 hinge, and replacing the non-IgG2 hinge with an IgG2 hinge.In certain embodiments, a modified heavy chain constant region comprisesa hinge of the IgG2 isotype (an “IgG2 hinge”) and a CH1, CH2 and CH3domain. In certain embodiments, a modified heavy chain constant regincomprises an IgG2 hinge and a CH1, CH2 and CH3 domain, wherein at leastone of the CH1, CH2 and CH3 domains is not of the IgG2 isotype. The IgG2hinge may be a wildtype IgG2 hinge, e.g., a wildtype human IgG2 hinge(e.g., ERKCCVECPPCPAPPVAG; SEQ ID NO: 208) or a variant thereof,provided that the IgG2 hinge retains the ability to confer to theantibody an altered activity relative to the same antibody thatcomprises a non-IgG2 hinge. In certain embodiments, an IgG2 hingevariant retains similar rigidity or stiffness to that of a wildtype IgG2hinge. The rigidity of a hinge can be determined, e.g., by computermodeling, electron microscopy, spectroscopy such as Nuclear MagneticResonance (NMR), X-ray crystallography (B-factors), or SedimentationVelocity Analytical ultracentrifugation (AUC) to measure or compare theradius of gyration of antibodies comprising the hinge. A hinge may havesimilar or higher rigidity relative to another hinge if an antibodycomprising the hinge has a value obtained from one of the testsdescribed in the previous sentence that differs from the value of thesame antibody with a different hinge, e.g., an IgG1 hinge, in less than5%, 10%, 25%, 50%, 75%, or 100%. A person of skill in the art would beable to determine from the tests whether a hinge has at least similarrigidity to that of another hinge by interpreting the results of thesetests. An exemplary human IgG2 hinge variant is an IgG2 hinge thatcomprises a substitution of one or more of the four cysteine residues(i.e., C219, C220, C226 and C229). A cysteine may be replaced by aserine. An exemplary IgG2 hinge is a human IgG2 hinge comprising a C219Smutation (e.g., ERKSCVECPPCPAPPVAG; SEQ ID NO: 209). Other IgG2 hingevariants that may be used include human IgG2 hinges comprising a C220,C226 and/or C229 substitution, e.g., a C220S, C226S or C229S mutation(which may be combined with a C219S mutation). An IgG2 hinge may also bean IgG2 hinge in which a portion of the hinge is that of another isotype(i.e., it is a chimeric hinge), provided that the rigidity of thechimeric hinge is at least similar to that of a wildtype IgG2 hinge. Forexample, an IgG2 hinge may be an IgG2 hinge in which the lower hinge (asdefined in Table 2) is of an IgG1 isotype, and is, e.g., a wildtype IgG1lower hinge. Additional IgG2 hinge mutations that may be used in an IgG2hinge include the SE (S267E), SELF (S267E/L328F), SDIE (S239D/I332E),SEFF and GASDALIE (G236A/S239D/A330L/I332E) mutations.

A “hybrid” or “chimeric” hinge is referred to as being of a specificisotype if more than half of the consecutive amino acids of the hingeare from that isotype. For example, a hinge having an upper and middlehinge of IgG2 and the lower hinge of IgG1 is considered to be an IgG2hinge.

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region that comprises an IgG2 hinge comprising one of thefollowing sequences:

(SEQ ID NO: 208) ERKCCVECPPCPAPPVAG; (SEQ ID NO: 209)ERKSCVECPPCPAPPVAG; (SEQ ID NO: 210) ERKCSVECPPCPAPPVAG;(SEQ ID NO: 211) ERKXCVECPPCPAPPVAG; (SEQ ID NO: 212)ERKCXVECPPCPAPPVAG; (SEQ ID NO: 213) ERKCCVECPPCPAPPVAGX;(SEQ ID NO: 214) ERKSCVECPPCPAPPVAGX; (SEQ ID NO: 215)ERKCSVECPPCPAPPVAGX; (SEQ ID NO: 216) ERKXCVECPPCPAPPVAGX;(SEQ ID NO: 217) ERKCXVECPPCPAPPVAGX; (SEQ ID NO: 218)ERKCCVECPPCPAPELLGG; (SEQ ID NO: 219) ERKSCVECPPCPAPELLGG;(SEQ ID NO: 220) ERKCCSVECPPCPAPELLGG; (SEQ ID NO: 221)ERKXCVECPPCPAPELLGG; (SEQ ID NO: 222) ERKCXVECPPCPAPELLGG;(SEQ ID NO: 223) ERKCCVECPPCPAPELLG; (SEQ ID NO: 224)ERKSCVECPPCPAPELLG; (SEQ ID NO: 225) ERKCCSVECPPCPAPELLG;(SEQ ID NO: 226) ERKXCVECPPCPAPELLG; (SEQ ID NO: 227)ERKCXVECPPCPAPELLG; (SEQ ID NO: 228) ERKCCVECPPCPAP; (SEQ ID NO: 229)ERKSCVECPPCPAP; (SEQ ID NO: 230) ERKCSVECPPCPAP; (SEQ ID NO: 231)ERKXCVECPPCPAP; or (SEQ ID NO: 232) ERKCXVECPPCPAP,

wherein X is any amino acid, except a cysteine,

or any of the above sequences, in which 1-5, 1-3, 1-2 or 1 amino acid isinserted between amino acid residues CVE and CPP. In certainembodiments, THT or GGG is inserted. In certain embodiments, 1, 1-2, or1-3 amino acids are inserted between the hinge and CH2 domain. Forexample, a glycine may be inserted between the hinge and CH2 domain.

In certain embodiments, the hinge comprises SEQ ID NO: 208, 209, 210,211, or 212, wherein 1, 2, 3 or all 4 amino acids P233, V234, A235 andG237 (corresponding to the C-terminal 4 amino acids “PVAG” (SEQ ID NO:233) are deleted or substituted with another amino acid, e.g., the aminoacids of the C-terminus of the IgG1 hinge (ELLG (SEQ ID NO: 234) orELLGG (SEQ ID NO: 235). In certain embodiments, the hinge comprises SEQID NO: 208, 209, 210, 211, or 212, wherein V234, A235 and G237 aredeleted or substituted with another amino acid. In certain embodiments,the hinge comprises SEQ ID NO: 208, 209, 210, 211, or 212, wherein A235and G237 are deleted or substituted with another amino acid. In certainembodiments, the hinge comprises SEQ ID NO: 208, 209, 210, 211, or 212,wherein G237 is deleted or substituted with another amino acid. Incertain embodiments, the hinge comprises SEQ ID NO: 447, 448, 449, 450,or 451, wherein V234 and A235 are deleted or substituted with anotheramino acid. Substitution of PVAG (SEQ ID NO: 233) in an IgG2 with thecorresponding amino acids of an IgG1 hinge, i.e., (ELLG (SEQ ID NO: 234)or ELLGG (SEQ ID NO: 235)) to obtain a hybrid hinge, e.g., shown above,that provides a hinge having the advantages of an IgG2 hinge and theeffector function of IgG1 hinges.

In certain embodiments, a modified heavy chain constant region comprisesa hinge that consists of or consists essentially of one of the sequencesshown above, e.g., any one of SEQ ID NOs: 208-232, and in certainembodiments, does not comprise additional hinge amino acid residues.

In certain embodiments, a modified heavy chain constant region comprisesa CH1 domain that is a wildtype CH1 domain of the IgG1 or IgG2 isotype(“IgG1 CH1 domain” or “IgG2 CH1 domain,” respectively). CH1 domains ofthe isotypes IgG3 and IgG4 (“IgG3 CH1 domain and “IgG2 CH1 domain,”respectively) may also be used. A CH1 domain may also be a variant of awildtype CH1 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3 orIgG4 CH1 domain. Exemplary variants of CH1 domains include A114C andT173C and/or C131, e.g., C131S.

In certain embodiments, a modified heavy chain constant region comprisesa CH2 domain that is a wildtype CH2 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG1 CH2 domain,” “IgG2 CH2 domain,” “IgG3 CH2 domain,”or “IgG4 CH2 domain,” respectively). A CH2 domain may also be a variantof a wildtype CH2 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3or IgG4 CH2 domain. Exemplary variants of CH2 domains include variantsthat modulate a biological activity of the Fc region of an antibody,such as ADCC or CDC or modulate the half-life of the antibody or itsstability. In one embodiment, the CH2 domain is a human IgG1 CH2 domainwith an A330S and P331S mutation, wherein the CH2 domain has reducedeffector function relative to the same CH2 mutation without themutations. Other mutations are further set forth herein elsewhere.

In certain embodiments, a modified heavy chain constant region comprisesa CH3 domain that is a wildtype CH3 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG1 CH3 domain,” “IgG2 CH3 domain,” “IgG3 CH3 domain,”or “IgG4 CH3 domain,” respectively). A CH3 domain may also be a variantof a wildtype CH3 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3or IgG4 CH3 domain. Exemplary variants of CH3 domains include variantsthat modulate a biological activity of the Fc region of an antibody,such as ADCC or CDC or modulate the half-life of the antibody or itsstability.

Generally, variants of the CH1, hinge, CH2 or CH3 domains may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and/or at most 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or comprisean amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to that of the corresponding wildtypedomain (CH1, hinge, CH2, or CH3 domain, respectively), provided that theheavy chain constant region comprising the specific variant retains thenecessary biological activity.

Table 3 sets forth exemplary human heavy chain constant regionscomprising a human CH1, hinge, CH2 and/or CH3 domains, wherein eachdomain is either a wildtype domain or a variant thereof that providesthe desired biological activity to the heavy chain constant region. Anunfilled cell in Table 3 indicates that the domain is present or not,and if present can be of any isotype, e.g., IgG1, IgG2, IgG3 or IgG4.For example, an antibody comprising the heavy chain constant region 1 inTable 3 is an antibody that comprises a heavy chain constant regioncomprising at least an IgG2 hinge, and which may also comprise a CH1,CH2 and/or CH3 domain, and if present, which CH1, CH2 and/or CH3 domainis of an IgG1, IgG2, IgG3 or IgG4 isotype. As another example forunderstanding Table 3, an antibody comprising a heavy chain constantregion 8 is an antibody comprising a heavy chain constant regioncomprising an IgG1 CH1 domain, and IgG2 hinge, an IgG1 CH2 domain, andwhich may or may not also comprise a CH3 domain, which if present, maybe of an IgG1, IgG2, IgG3 or IgG4 isotype.

TABLE 3 Exemplary configurations of human heavy chain constant regionsMHCCR* CH1 Hinge CH2 CH3 1 IgG2 2 IgG1 IgG2 3 IgG2 IgG2 4 IgG2 IgG1 5IgG2 IgG2 6 IgG2 IgG1 7 IgG2 IgG2 8 IgG1 IgG2 IgG1 9 IgG1 IgG2 IgG2 10IgG2 IgG2 IgG1 11 IgG2 IgG2 IgG2 12 IgG1 IgG2 IgG1 13 IgG1 IgG2 IgG2 14IgG2 IgG2 IgG1 15 IgG2 IgG2 IgG2 16 IgG2 IgG1 IgG1 17 IgG2 IgG1 IgG2 18IgG2 IgG2 IgG1 19 IgG2 IgG2 IgG2 20 IgG1 IgG2 IgG1 IgG1 21 IgG1 IgG2IgG1 IgG2 22 IgG1 IgG2 IgG2 IgG1 23 IgG1 IgG2 IgG2 IgG2 24 IgG2 IgG2IgG1 IgG1 25 IgG2 IgG2 IgG1 IgG2 26 IgG2 IgG2 IgG2 IgG1 27 IgG2 IgG2IgG2 IgG2 *Modified heavy chain constant region

In certain embodiments, an anti-OX40 antibody comprises a heavy chainconstant region shown in Table 3 and may have altered activity relativeto the same antibody comprising a heavy chain constant region that doesnot comprise that specific heavy chain constant region. In certainembodiments, an antibody comprising a heavy chain constant region shownin Table 3 or 4 may have an altered activity relative to the sameantibody comprising a heavy chain constant region that does not comprisean IgG2 hinge or the same IgG2 hinge. In certain embodiments, anantibody comprising a heavy chain constant region shown in Table 3 or 4may have an altered activity relative to the same antibody comprising aheavy chain constant region that comprises a non-IgG2 hinge, andcomprises, e.g., an IgG1, IgG3 or IgG4 hinge. In certain embodiments, anantibody comprising a heavy chain constant region shown in Table 3 or 4may have an altered activity relative to the same antibody comprising aheavy chain constant region that does not comprise one or more of thesame CH1, hinge, CH2 or CH3 domain. For example, in certain embodiments,an antibody comprising a heavy chain constant region shown in Table 3 or4 may have an altered activity relative to the same antibody comprisinga heavy chain constant region that does not comprise an IgG2 hinge and aCH1, CH2 and/or CH3 domain of the specific isotype. For example, anantibody comprising a heavy chain constant region 22 shown in Table 3,may have an altered activity relative to (i) the same antibodycomprising a heavy chain constant region that does not comprise an IgG2hinge, and comprises, e.g., a non-IgG2 hinge (e.g., an IgG1, IgG3 orIgG4 hinge); (ii) the same antibody comprising a heavy chain constantregion that does not comprise an IgG2 hinge and an IgG1 CH1, andcomprises, e.g., a non-IgG2 hinge and/or a non-IgG1 CH1; (iii) the sameantibody comprising a heavy chain constant region that does not comprisean IgG2 hinge and an IgG2 CH2, and comprises, e.g., a non-IgG2 hingeand/or a non-IgG2 CH2; (iv) the same antibody comprising a heavy chainconstant region that does not comprise an IgG2 hinge and an IgG1 CH3,and comprises, e.g., a non-IgG2 hinge and/or a non-IgG1 CH3; (v) thesame antibody comprising a heavy chain constant region that does notcomprise an IgG2 hinge, an IgG1 CH1 and an IgG2 CH2, and comprises,e.g., a non-IgG2 hinge and/or a non-IgG1 CH1 and/or a non-IgG2 CH2; (vi)the same antibody comprising a heavy chain constant region that does notcomprise an IgG2 hinge, an IgG1 CH1 and an IgG1 CH3, and comprises,e.g., a non-IgG2 hinge and/or a non-IgG1 CH1 and/or a non-IgG1 CH3;(vii) the same antibody comprising a heavy chain constant region thatdoes not comprise an IgG2 hinge, an IgG2 CH2 and an IgG1 CH3, andcomprises, e.g., a non-IgG2 hinge and/or a non-IgG2 CH and/or a non-IgG1CH3; (viii) or the same antibody comprising a heavy chain constantregion that does not comprise an IgG2 hinge, an IgG1 CH1, IgG2 CH2 andan IgG1 CH3, and comprises, e.g., a non-IgG2 hinge and/or a non-IgG1 CH1and/or a non-IgG2 CH2 and/or a non-IgG1 CH3.

Exemplary modified heavy chain constant regions that may be linked toanti-OX40 variable regions, e.g., the variable regions described herein,are provided in Table 4, which sets forth the identity of each of thedomains.

TABLE 4 Exemplary modified heavy chain constant regions Modified SEQ IDheavy chain NO of constant whole region CH1 Hinge CH2 CH3 MHCCRIgG1-IgG2- IgG1 IgG2/IgG1 IgG1 IgG1 wildtype SEQ ID IgG1f wildtype SEQID wildtype SEQ ID NO: 244 SEQ ID NO: 240 SEQ ID NO: 204 NO: 202 NO: 204IgG1-IgG2- IgG1 IgG2 wildtype IgG1 IgG1 wildtype SEQ ID IgG1f2 wildtypeSEQ ID wildtype SEQ ID NO: 245 SEQ ID NO: 238 SEQ ID NO: 206 NO: 202 NO:204 IgG1- IgG1 IgG2C219S/IgG1 IgG1 IgG1 wildtype SEQ ID IgG2CS- wildtypeSEQ ID wildtype SEQ ID NO: 246 IgG1f SEQ ID NO: 241 SEQ ID NO: 206 NO:202 NO: 204 IgG1- IgG1 IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG2CS-wildtype SEQ ID wildtype SEQ ID NO: 247 IgG1f2 SEQ ID NO: 239 SEQ ID NO:206 NO: 202 NO: 204 IgG2-IgG1f IgG2 IgG2/IgG1 IgG1 IgG1 wildtype SEQ IDwildtype SEQ ID wildtype SEQ ID NO: 248 SEQ ID NO: 240 SEQ ID NO: 206NO: 203 NO: 204 IgG2-IgG1f2 IgG2 IgG2 wildtype IgG1 IgG1 wildtype SEQ IDwildtype SEQ ID wildtype SEQ ID NO: 249 SEQ ID NO: 238 SEQ ID NO: 206NO: 203 NO: 204 IgG2CS- IgG2 IgG2C219S/IgG1 IgG1 IgG1 wildtype SEQ IDIgG1f wildtype SEQ ID wildtype SEQ ID NO: 250 SEQ ID NO: 241 SEQ ID NO:206 NO: 203 NO: 204 IgG2CS- IgG2 IgG2 C219S IgG1 IgG1 wildtype SEQ IDIgG1f2 wildtype SEQ ID wildtype SEQ ID NO: 251 SEQ ID NO: 239 SEQ ID NO:206 NO: 203 NO: 204 IgG1-IgG2- IgG1 IgG2 wildtype IgG1 IgG1 wildtype SEQID IgG1.1f wildtype SEQ ID A330S/P331S SEQ ID NO: 252 SEQ ID NO: 238 SEQID NO: 206 NO: 202 NO: 243 IgG1- IgG1 IgG2 C219S IgG1 IgG1 wildtype SEQID IgG2CS- wildtype SEQ ID A330S/P331S SEQ ID NO: 253 IgG1.1f SEQ ID NO:239 SEQ ID NO: 206 NO: 202 NO: 243 IgG2-IgG1.1f IgG2 IgG2 wildtype IgG1IgG1 wildtype SEQ ID wildtype SEQ ID A330S/P331S SEQ ID NO: 254 SEQ IDNO: 238 SEQ ID NO: 206 NO: 203 NO: 243 IgG2CS- IgG2 IgG2 C219S IgG1 IgG1wildtype SEQ ID IgG1.1f wildtype SEQ ID A330S/P331S SEQ ID NO: 255 SEQID NO: 239 SEQ ID NO: 206 NO: 203 NO: 243

Additional exemplary modified heavy chain constant regions are providedin Table 5.

TABLE 5 SEQ ID NO of constant Constructs region Description IgG1f 256wild type IgG1f IgG1.1f 257 standard inert IgG1.1f IgG2.3 258 IgG2A-form (C219S) IgG2.5 259 IgG2 B-form (C131S) IgG2.3G1-KH 260 CH1, upperhinge and lower hinge/ upper CH2 of IgG2.3, all else IgG1f IgG2.5G1-KH261 CH1, upper hinge and lower hinge/ upper CH2 of IgG2.5, all elseIgG1f IgG2.3G1-AY 262 CH1 and upper hinge of IgG2.3, all else IgG1fIgG2.5G1-AY 263 CH1 and upper hinge of IgG2.5, all else IgG1fIgG2.3G1.1f-KH 264 CH1, upper hinge and lower hinge/ upper CH2 ofIgG2.3, all else IgG1.1f IgG2.5G1.1f-KH 265 CH1 upper hinge and lowerhinge/ upper CH2 of IgG2.5, all else IgG1.1f IgG2.5G1-V27 266IgG2-B-form variant IgG2.3G1-V27 297 hHC-1gG2-C219S/hHC-IgG1f - S267E

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region comprising an IgG2 hinge comprising any one of SEQID NOs: 238, 239, 240, 241, and 208-232 or a variant thereof, such as anIgG2 hinge comprising an amino acid sequence that (i) differs from anyone of SEQ ID NOs: 238, 239, 240, 241, and 208-232 in 1, 2, 3, 4 or 5amino acids substitutions, additions or deletions; (ii) differs from anyone of SEQ ID NOs: 238, 239, 240, 241, and 208-232 in at most 5, 4, 3,2, or 1 amino acids substitutions, additions or deletions; (iii) differsfrom any one of SEQ ID NOs: 238, 239, 240, 241, and 208-232 in 1-5, 1-3,1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or(iv) comprises an amino acid sequence that is at least about 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:238, 239, 240, 241, and 208-232, wherein in any of (i)-(iv), an aminoacid substitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region provides an altered activity to an anti-OX40antibody relative to another heavy chain constant region, e.g., a heavychain constant region that comprises a non-IgG2 hinge or relative to thesame modified heavy chain constant region that comprises a non-IgG2hinge.

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region comprising an IgG1 CH1 domain comprising SEQ IDNO: 202 or an IgG2 CH1 domain comprising SEQ ID NO: 203, or a variant ofSEQ ID NO: 202 or 203, which variant (i) differs from SEQ ID NO: 202 or203 in 1, 2, 3, 4 or 5 amino acids substitutions, additions ordeletions; (ii) differs from SEQ ID NO: 202 or 203 in at most 5, 4, 3,2, or 1 amino acids substitutions, additions or deletions; (iii) differsfrom SEQ ID NO: 202 or 203 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acidssubstitutions, additions or deletions and/or (iv) comprises an aminoacid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 202 or 203, wherein in any of(i)-(iv), an amino acid substitution may be a conservative amino acidsubstitution or a non-conservative amino acid substitution; and whereinthe anti-OX40 antibody comprising a modified heavy chain constant regionmay have an altered activity relative to that of the anti-OX40 antibodybut with another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or relative to the samemodified heavy chain constant region that comprises a non-IgG2 hinge.

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region comprising an IgG1 CH2 domain comprising SEQ IDNO: 204 or 298, or a variant of SEQ ID NO: 204 or 298, which variant (i)differs from SEQ ID NO: 204 or 298 in 1, 2, 3, 4 or 5 amino acidssubstitutions, additions or deletions; (ii) differs from SEQ ID NO: 204or 298 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additionsor deletions; (iii) differs from SEQ ID NO: 204 or 298 in 1-5, 1-3, 1-2,2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv)comprises an amino acid sequence that is at least about 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 204 or 298,wherein in any of (i)-(iv), an amino acid substitution may be aconservative amino acid substitution or a non-conservative amino acidsubstitution; and wherein the modified heavy chain constant region mayprovide an altered activity to an anti-OX40 antibody relative to that ofanother heavy chain constant region, e.g., a heavy chain constant regionthat comprises a non-IgG2 hinge or relative to the same modified heavychain constant region that comprises a non-IgG2 hinge.

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region comprising an IgG1 CH3 domain comprising SEQ IDNO: 206, or a variant of SEQ ID NO: 206, which variant (i) differs fromSEQ ID NO: 206 in 1, 2, 3, 4 or 5 amino acids substitutions, additionsor deletions; (ii) differs from SEQ ID NO: 206 in at most 5, 4, 3, 2, or1 amino acids substitutions, additions or deletions; (iii) differs fromSEQ ID NO: 206 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions,additions or deletions and/or (iv) comprises an amino acid sequence thatis at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 206, wherein in any of (i)-(iv), an amino acidsubstitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region may provide an altered activity relative to thatof another heavy chain constant region, e.g., a heavy chain constantregion that comprises a non-IgG2 hinge or relative to the same modifiedheavy chain constant region that comprises a non-IgG2 hinge.

Modified heavy chain constant regions may also comprise a combination ofthe CH1, hinge, CH2 and CH3 domains described above.

In certain embodiments, an anti-OX40 antibody comprises a modified heavychain constant region comprising any one of SEQ ID NOs: 244-281, or avariant of any one of SEQ ID NOs: 244-281, which variant (i) differsfrom any one of SEQ ID NOs: 244-281 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore amino acids substitutions, additions or deletions; (ii) differsfrom any one of SEQ ID NOs: 244-281 in at most 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acids substitutions, additions or deletions; (iii) differsfrom any one of SEQ ID NOs: 244-281 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or5-10 amino acids substitutions, additions or deletions and/or (iv)comprises an amino acid sequence that is at least about 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:244-281, wherein in any of (i)-(iv), an amino acid substitution may be aconservative amino acid substitution or a non-conservative amino acidsubstitution; and wherein the modified heavy chain constant region mayprovide an altered activity relative to that of another heavy chainconstant region, e.g., a heavy chain constant region that comprises anon-IgG2 hinge or relative to the same modified heavy chain constantregion that comprises a non-IgG2 hinge.

Modified heavy chain constant regions may have (i) similar, reduced orincreased effector function (e.g., binding to an FcγR) relative to awildtype heavy chain constant region and or (ii) similar, reduced orincreased half-life (or binding to the FcRn receptor) relative to awildtype heavy chain constant region.

III. Antibodies Having Particular Germline Sequences

In certain embodiments, anti-OX40 antibodies described herein comprise aheavy chain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

As discussed in the Examples of the present disclosure, human antibodiesspecific for OX40 have been prepared that comprise a heavy chainvariable region that is the product of or derived from human germline VH1-08 gene, VH 6-6 gene, VH 5-51 gene, VH 3-9 gene, VH DP44 gene, VH3-30.3 gene, VH 3-10 gene, and/or VH 3-13 gene. Accordingly, providedherein are isolated monoclonal antibodies specific for human OX40, orantigen-binding portions thereof, comprising a heavy chain variableregion that is the product of or derived from a human VH germline geneselected from the group consisting of: VH 1-08 gene, VH 6-6 gene, VH5-51 gene, VH 3-9 gene, VH DP44 gene, VH 3-30.3 gene, VH 3-10 gene, andVH 3-13 gene.

Human antibodies specific for OX40 have been prepared that comprise alight chain variable region that is the product of or derived from humangermline VK L5 gene, VK L6 gene, VK L15 gene, VK A27 gene, and/or VKO14/O4 gene. Accordingly, provide herein are isolated monoclonalantibodies, or antigen-binding portions thereof, comprising a lightchain variable region that is the product of or derived from a human VKgermline gene selected from the group consisting of: VK L5 gene, VK L6gene, VK L15 gene, VK A27 gene, and VK O14/O4 gene.

Preferred antibodies described herein are those comprising a heavy chainvariable region that is the product of or derived from one of theabove-listed human germline VH genes and also comprising a light chainvariable region that is the product of or derived from one of theabove-listed human germline VK genes.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

IV. Homologous Antibodies

Provided herein are antibodies having heavy and light chain variableregions comprising amino acid sequences that are homologous to the aminoacid sequences of the preferred antibodies described herein, and whereinthe antibodies retain the desired functional properties of the anti-OX40antibodies described herein.

For example, an isolated anti-OX40 antibody, or antigen binding portionthereof, may comprise a heavy chain variable region and a light chainvariable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:318, 17, 28, 37, 48, 57, 65, 73, 84, and 93, or comprises 1, 2, 3, 4, 5,1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acid changes(i.e., amino acid substitutions, additions or deletions) relative to anamino acid sequence selected from the group consisting of SEQ ID NOs:318, 17, 28, 37, 48, 57, 65, 73, 84, and 93;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:18, 29, 30, 38, 49, 50, 58, 66, 74, 85, 86, and 94, or comprises 1, 2,3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acidchanges (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66, 74, 85, 86, and 94;

(c) the antibody specifically binds to OX40, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, or all of the followingfunctional properties:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

The antibody can be, for example, a human antibody, a humanized antibodyor a chimeric antibody.

In certain embodiments, the anti-OX40 antibody, or antigen bindingportion thereof, may comprise a heavy chain and a light chain, wherein:

(a) the heavy chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125,or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25,or 1-50 amino acid changes (i.e., amino acid substitutions, additions ordeletions) relative to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 124 and 125;

(b) the light chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or comprises1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 aminoacid changes (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, and 122;

(c) the antibody specifically binds to OX40, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, or all of the followingfunctional properties:

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g., as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

Also provided are anti-OX40 antibodies comprising a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3 that differs from thecorresponding CDR of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1, in 1, 2, 3, 4, 5, 1-2, 1-3,1-4, or 1-5 amino acid changes (i.e., amino acid substitutions,additions or deletions). In certain embodiments, the antibody comprises1-5 amino acid changes in each of 1, 2, 3, 4, 5 or 6 of the CDRsrelative to the corresponding sequence in 3F4, 14B6-1, 14B6-2, 23H3,6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1. In certainembodiments, the antibody comprises at total of 1-5 amino acid changesacross all CDRs relative to the CDRs in 3F4, 14B6-1, 14B6-2, 23H3,6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1.

Antibodies having sequences with homology to those of 3F4, 14B6 (14B6-1and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1and 14A2-2), and 20C1, e.g., the V_(H) and V_(L) regions of SEQ ID NOs:17 and 18; 28 and 29; 28 and 30; 37 and 38; 48 and 49; 48 and 50; 57 and58; 65 and 66; 73 and 74; 84 and 85; 84 and 86; 93 and 94, respectively,or heavy and light chains of SEQ ID NOs: 95 and 96; 97 and 98; 99 and100; 101 and 102; 103 and 104; 105 and 106; 107 and 108; 109 and 110;111 and 112; 113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and122; 123 and 116; 124 and 116; and 125 and 116, respectively, or CDRscan be obtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 17, 28, 37,48, 57, 65, 73, 84, and 93 and/or SEQ ID NOs: 18, 29, 30, 38, 49, 50,58, 66, 74, 85, 86, and 94 or SEQ ID NOs: 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125 and/or SEQ IDNOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and122, followed by testing of the encoded altered antibody for retainedfunction (i.e., the functions set forth in (1) through (7) above) usingthe functional assays described herein.

V. Antibodies with Conservative Modifications

Anti-OX40 antibodies provided herein may comprise a heavy chain variableregion comprising CDR1, CDR2 and CDR3 sequences and a light chainvariable region comprising CDR1, CDR2 and CDR3 sequences, wherein one ormore of these CDR sequences comprise specified amino acid sequencesbased on the antibodies described herein (e.g., 3F4, 14B6-1, 14B6-2,23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1), orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties of the anti-OX40 antibodies describedherein. Accordingly, the anti-OX40 antibody, or antigen binding portionthereof, may comprise a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77,        and 89, and conservative modifications thereof, e.g., 1, 2, 3,        4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid        substitutions;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72,        80, 83, and 92, and conservative modifications thereof, e.g., 1,        2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid        substitutions;    -   (c) the antibody specifically binds to OX40, and    -   (d) the antibody exhibits 1, 2, 3, 4, 5, 6, or all of the        following functional properties:        -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10            nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by            Biacore;        -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀            of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured            by FACS;        -   (3) binding to cynomolgus OX40, e.g., binding to membrane            bound cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less            (e.g., 0.01 nM to 10 nM), e.g., as measured by FACS;        -   (4) inducing or enhancing T cell activation, as evidenced            by (i) increased IL-2 and/or IFN-γ production in            OX40-expressing T cells and/or (ii) enhanced T cell            proliferation;        -   (5) inhibiting the binding of OX40 ligand to OX40, e.g.,            with an EC₅₀ of 1 nM or less as measured by FACS, e.g., in            an assay with hOX40-293 cells;        -   (6) binding to an epitope on the extracellular portion of            mature human OX40 (SEQ ID NO: 2), e.g., an epitope within            the region

(SEQ ID NO: 178) DVVSSKPCKPCTWCNLR or (SEQ ID NO: 179)DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK;

-   -   -   (7) competing for binding to human OX40 with 3F4, 14B6-1,            14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;        -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,            14A2-1, and 14A2-2.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88,and 317, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5,1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; and thelight chain variable region CDR2 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions. In another preferredembodiment, the heavy chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions; and the light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 14, 22,25, 34, 42, 45, 54, 62, 70, 78, 81, and 90, and conservativemodifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5conservative amino acid substitutions.

In various embodiments, the antibodies can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

Conservative amino acid substitutions may also be made in portions ofthe antibodies other than, or in addition to, the CDRs. For example,conservative amino acid modifications may be made in a framework regionor in the Fc region. A variable region or a heavy or light chain maycomprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 conservative amino acid substitutions relative to the anti-OX40antibody sequences provided herein. In certain embodiments, theanti-OX40 antibody comprises a combination of conservative andnon-conservative amino acid modification.

VI. Competing Antibodies and Same Epitope Binding Antibodies

Also provided herein are antibodies that compete for binding to OX40with the anti-OX40 antibodies described herein (e.g., antibodies 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1). Such competing antibodies can be identified based on theirability to competitively inhibit binding to OX40 of one or more ofmonoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,8B11, 20B3, 14A2-1, 14A2-2, and 20C1 in standard OX40 binding assays.For example, standard ELISA assays or competitive ELISA assays can beused in which a recombinant human OX40 protein is immobilized on aplate, various concentrations of unlabeled first antibody are added, theplate is washed, labeled second antibody is added, washed, and theamount of bound label is measured. If the increasing concentration ofthe unlabeled (first) antibody (also referred to as the “blockingantibody”) inhibits the binding of the labeled (second) antibody, thefirst antibody is said to inhibit the binding of the second antibody tothe target on the plate, or is said to compete with the binding of thesecond antibody. Additionally or alternatively, BIACORE® SPR analysiscan be used to assess the ability of the antibodies to compete. Theability of a test antibody to inhibit the binding of an anti-OX40antibody described herein to OX40 demonstrates that the test antibodycan compete with the antibody for binding to OX40.

Accordingly, provided herein are anti-OX40 antibodies that inhibit thebinding of the anti-OX40 antibodies described herein to OX40 on cells,e.g., activated T cells, by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%, by using, e.g., FACS as described in the Examples.

In other embodiments, provided herein are anti-OX40 antibodies whichbind to the same epitope as one or more of the anti-OX40 antibodiesdescribed herein (e.g., antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1,6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1), as determined usingart-recognized epitope mapping techniques, such as those describedbelow.

Art-recognized epitope mapping techniques include, for example,structural methods, such as X-ray crystal structure determination (e.g.,WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)spectroscopy, including NMR determination of the H-D exchange rates oflabile amide hydrogens in OX40 when free and when bound in a complexwith an antibody of interest (Zinn-Justin et al. Biochemistry 1992;31:11335-47; Zinn-Justin et al. Biochemistry 1993; 32, 6884-91).

For X-ray crystallography, crystallization may be accomplished using anyknown method in the art (e.g. Giege et al. Acta Crystallogr 1994;D50:339-50; McPherson, Eur J Biochem 1990; 189:1-23), includingmicrobatch (e.g. Chayen, Structure 19976; 5:1269-74), hanging-drop vapordiffusion (e.g. McPherson, J Biol Chem 1976; 251:6300-3), seeding anddialysis. It is desirable to use a protein preparation having aconcentration of at least about 1 mg/mL and preferably about 10 mg/mL toabout 20 mg/mL. Crystallization may be best achieved in a precipitantsolution containing polyethylene glycol 1000-20,000 (PEG; averagemolecular weight ranging from about 1000 to about 20,000 Da), preferablyabout 5000 to about 7000 Da, more preferably about 6000 Da, withconcentrations ranging from about 10% to about 30% (w/v). It may also bedesirable to include a protein stabilizing agent, e.g., glycerol at aconcentration ranging from about 0.5% to about 20%. A suitable salt,such as sodium chloride, lithium chloride or sodium citrate may also bedesirable in the precipitant solution, preferably in a concentrationranging from about 1 mM to about 1000 mM. The precipitant is preferablybuffered to a pH of from about 3.0 to about 5.0, preferably about 4.0.Specific buffers useful in the precipitant solution may vary and arewell-known in the art (Scopes, Protein Purification: Principles andPractice, Third ed., (1994) Springer-Verlag, New York). Examples of suchbuffers include, but are not limited to, HEPES, Tris, MES and acetate.Crystals may be grow at a wide range of temperatures, including 2° C.,4° C., 8° C. and 26° C. Antibody:antigen crystals may be studied usingwell-known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see e.g. Blundell & Johnson, Meth. Enzymol.1985; 114 & 115, H. W. Wyckoff et al., eds., Academic Press; U.S. PatentApplication Publication No. 2004/0014194), and BUSTER (Bricogne, ActaCryst 1993; D49:37-60; Bricogne, Meth Enzymol 1997; 276A:361-423; Carter& Sweet, eds.; Roversi et al., Acta Cryst. 2000; D56:1313-23).

Other epitope mapping methods monitor the binding of the antibody toantigen fragments or mutated variations of the antigen where loss ofbinding due to a modification of an amino acid residue within theantigen sequence is often considered an indication of an epitopecomponent. One such method is alanine scanning mutagenesis, asdescribed, e.g., by Cunningham and Wells, Science 1989; 244:1081-5.Another suitable method is deep mutational scanning (see, e.g., Araya etal., Trends in Biotechnology 2011; 29:435-42; Forsyth et al., mAbs 2013;5:523-32).

Additionally or alternatively, computational combinatorial methods forepitope mapping, including the mapping of conformational discontinuousepitopes, can be used.

Additionally or alternatively, epitope mapping can be achieved bytesting binding of an antibody to peptides comprising fragments of OX40,e.g., non-denatured or denatured fragments. A series of overlappingpeptides encompassing the sequence of OX40 (e.g., human OX40) may besynthesized and screened for binding, e.g., in a direct ELISA, acompetitive ELISA (where the peptide is assessed for its ability toprevent binding of an antibody to OX40 bound to a well of a microtiterplate) or on a chip. Other methods rely on the ability of an antibody ofinterest to affinity isolate specific short peptides (either in nativethree dimensional form or in denatured form) from combinatorial phagedisplay peptide libraries. The peptides are then regarded as leads forthe definition of the epitope recognized by the antibody used to screenthe peptide library.

Epitopes also can be identified by MS-based protein footprinting, suchas Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and FastPhotochemical Oxidation of Proteins (FPOP). HDX-MS may be conducted, forexample, as described in the Examples herein and by Wei et al., DrugDiscovery Today 2014; 19:95. FPOP may be conducted, for example, asdescribed by Hambley et al. (J American Soc Mass Spectrometry 2005;16:2057).

Antibodies that compete for binding with the anti-OX40 antibodiesdescribed herein may be produced and identified using art-known methods.For example, mice may be immunized with human OX40 as described herein,hybridomas produced, and the resulting monoclonal antibodies screenedfor the ability to compete with an antibody described herein for bindingto OX40 using the methods described above.

Antibodies that bind to the same epitope as the anti-OX40 antibodiesdescribed herein may be produced by immunizing mice with a smallerfragment of OX40 containing the epitope to which the antibody binds. Theepitope or region comprising the epitope can be identified using themethods described above. Alternatively, the method of Jespers et al.(Biotechnology 1994; 12:899) may be used to guide the selection ofantibodies recognizing the same epitope and therefore exhibiting similarproperties to the anti-OX40 antibodies described herein. For example,using phage display, first the heavy chain of the anti-OX40 antibody ispaired with a repertoire of (preferably human) light chains to select aOX40-binding antibody, and then the new light chain is paired with arepertoire of (preferably human) heavy chains to select a (preferablyhuman) OX40-binding antibody recognizing the same epitope or epitoperegion on OX40 as an anti-OX40 antibody described herein. Alternativelyvariants of an antibody described herein can be obtained by mutagenesisof cDNA encoding the heavy and light chains of the antibody.

In some embodiments, provided herein are antibodies which bind to all ora portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178),corresponding to amino acid residues 46-62 of mature human OX40 (SEQ IDNO: 2), as determined by the methods in the Examples.

In certain embodiments, the anti-OX40 antibodies described herein thatbind to all or a portion of the sequenceSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182), as determined by themethods in the Examples.

In other embodiments, the anti-OX40 antibodies described herein thatbind to all or a portion of the sequence PCKPCTWCNLR (SEQ ID NO: 183),as determined by the methods in the Examples.

In yet other embodiments, the anti-OX40 antibodies that bind to all or aportion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) further bindto all or a portion of the sequence QLCTATQDTVCR (SEQ ID NO: 184), asdetermined by the methods in the Examples.

In additional embodiments, the anti-OX40 antibodies described hereinthat bind to all or a portion of the sequence SQNTVCRPCGPGFYN (SEQ IDNO: 185), as determined by the methods in the Examples.

In additional embodiments, the anti-OX40 antibody binds within theregion DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179),corresponding to amino acid residues 89-124 of mature human OX40 (SEQ IDNO: 2), as determined by the methods in the Examples.

VII. Engineered and Modified Antibodies VH and VL Regions

Also provided herein are engineered and modified antibodies that can beprepared using an antibody having one or more of the V_(H) and/or V_(L)sequences disclosed herein as starting material to engineer a modifiedantibody, which modified antibody may have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i.e., V_(H) and/orV_(L)), for example within one or more CDR regions and/or within one ormore framework regions. Additionally or alternatively, an antibody canbe engineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificreference antibodies by constructing expression vectors that include CDRsequences from the specific reference antibody grafted onto frameworksequences from a different antibody with different properties (see,e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al.(1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad.See. U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)

Accordingly, another embodiment pertains to a monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences comprising an aminoacid sequence selected from the group consisting of SEQ ID SEQ ID NOs:11-13; 19-21; 31-33; 39-41; 51-53; 59-61; 67-69; 74-77; and 87-89,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 14-16; 22-24; 25-27; 34-36; 42-44;45-47; 54-56; 62-64; 70-72; 78-80; 81-83; and 90-92, respectively. Thus,such antibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,14A2-1, 14A2-2, and 20C1, yet may contain different framework sequences.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Preferred framework sequences for use in the antibodies described hereinare those that are structurally similar to the framework sequences usedby antibodies described herein. The V_(H) CDR1, 2 and 3 sequences, andthe V_(L) CDR1, 2 and 3 sequences, can be grafted onto framework regionsthat have the identical sequence as that found in the germlineimmunoglobulin gene from which the framework sequence derive, or the CDRsequences can be grafted onto framework regions that contain up to 20,preferably conservative, amino acid substitutions as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Engineered antibodies described herein include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

Another type of variable region modification is to mutate amino acidresidues within the CDR regions to improve one or more bindingproperties (e.g., affinity) of the antibody of interest. Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Preferably conservativemodifications (as discussed above) are introduced. The mutations may beamino acid additions, deletions, or preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, also provided herein are anti-OX40 monoclonal antibodies,or antigen binding portions thereof, comprising a heavy chain variableregion comprising: (a) a V_(H) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 11, 19, 31,39, 51, 59, 67, 75, and 87, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87; (b) aV_(H) CDR2 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88, and317, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 12, 20, 32, 40, 52, 60, 68, 76, and 88; (c) a V_(H) CDR3 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 13, 21, 33, 41, 53,61, 69, 77, and 89; (d) a V_(L) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 22, 25,34, 42, 45, 54, 62, 70, 78, 81, and 90, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70,78, 81, and 90; (e) a V_(L) CDR2 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 15, 23, 26,35, 43, 46, 55, 63, 71, 79, 82, and 91, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71,79, 82, and 91; and (f) a V_(L) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 16, 24, 27,36, 44, 47, 56, 64, 72, 80, 83, and 92, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72,80, 83, and 92.

Methionine residues in CDRs of antibodies can be oxidized, resulting inpotential chemical degradation and consequent reduction in potency ofthe antibody. Accordingly, one or more methionine residues in the heavyand/or light chain CDRs of the anti-OX40 antibodies described herein maybe replaced with amino acid residues that do not undergo oxidativedegradation.

Similarly, deamidation sites may be removed from the antibodies,particularly in the CDRs.

Potential glycosylation sites within the antigen binding domain arepreferably eliminated to prevent glycosylation that may interfere withantigen binding. See, e.g., U.S. Pat. No. 5,714,350.

Targeted Antigen Binding

In various embodiments, the antibodies described herein are modified toselectively block antigen binding in tissues and environments whereantigen binding would be detrimental, but allow antigen binding where itwould be beneficial. In one embodiment, a blocking peptide “mask” isgenerated that specifically binds to the antigen binding surface of theantibody and interferes with antigen binding, which mask is linked toeach of the binding arms of the antibody by a peptidase cleavablelinker. See, e.g., U.S. Pat. No. 8,518,404 to CytomX. Such constructsare useful for treatment of cancers in which protease levels are greatlyincreased in the tumor microenvironment compared with non-tumor tissues.Selective cleavage of the cleavable linker in the tumor microenvironmentallows disassociation of the masking/blocking peptide, enabling antigenbinding selectively in the tumor, rather than in peripheral tissues inwhich antigen binding might cause unwanted side effects.

Alternatively, in a related embodiment, a bivalent binding compound(“masking ligand”) comprising two antigen binding domains is developedthat binds to both antigen binding surfaces of the (bivalent) antibodyand interfere with antigen binding, in which the two binding domainsmasks are linked to each other (but not the antibody) by a cleavablelinker, for example cleavable by a peptidase. See, e.g., Int'l Pat. App.Pub. No. WO 2010/077643 to Tegopharm Corp. Masking ligands may comprise,or be derived from, the antigen to which the antibody is intended tobind, or may be independently generated. Such masking ligands are usefulfor treatment of cancers in which protease levels are greatly increasedin the tumor microenvironment compared with non-tumor tissues. Selectivecleavage of the cleavable linker in the tumor microenvironment allowsdisassociation of the two binding domains from each other, reducing theavidity for the antigen-binding surfaces of the antibody. The resultingdissociation of the masking ligand from the antibody enables antigenbinding selectively in the tumor, rather than in peripheral tissues inwhich antigen binding might cause unwanted side effects.

Fcs and Modified Fcs

In addition to the activity of a therapeutic antibody arising frombinding of the antigen binding domain to the antigen (e.g. blocking of acognate ligand or receptor protein in the case of antagonist antibodies,or induced signaling in the case of agonist antibodies), the Fc portionof the antibody interact with the immune system generally in complexways to elicit any number of biological effects. Effector functions,such as the Fc region of an immunoglobulin, are responsible for manyimportant antibody functions, such as antigen-dependent cellularcytotoxicity (ADCC), complement dependent cytotoxicity (CDC), andantibody-dependent cell-mediated phagocytosis (ADCP), result in killingof target cells, albeit by different mechanisms. There are five majorclasses, or isotypes, of heavy chain constant region (IgA, IgG, IgD,IgE, IgM), each with characteristic effector functions. These isotypescan be further subdivided into subclasses, for example, IgG is separatedinto four subclasses known as IgG1, IgG2, IgG3, and IgG4. IgG moleculesinteract with three classes of Fcγ receptors (FcγR) specific for the IgGclass of antibody, namely FcγRI, FcγRII, and FcγRIII. The importantsequences for the binding of IgG to the FcγR receptors have beenreported to be located in the CH2 and CH3 domains. The serum half-lifeof an antibody is influenced by the ability of that antibody to bind tothe neonatal Fc receptor (FcRn).

Anti-OX40 antibodies described herein may comprise the variable domainsof the invention combined with constant domains comprising different Fcregions, selected based on the biological activities (if any) of theantibody for the intended use. Salfeld (2007) Nat. Biotechnol. 25:1369.Human IgGs, for example, can be classified into four subclasses, IgG1,IgG2, IgG3, and IgG4, and each these of these comprises an Fc regionhaving a unique profile for binding to one or more of Fcγ receptors(activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIAand FcγRIIIB (CD16) and inhibiting receptor FcγRIIB), and for the firstcomponent of complement (C1q). Human IgG1 and IgG3 bind to all Fcγreceptors; IgG2 binds to FcγRIIA_(H131), and with lower affinity toFcγRIIA_(R131) FcγRIIIA_(V158); IgG4 binds to FcγRI, FcγRIIA, FcγRIIB,FcγRIIC, and FcγRIIIA_(V158); and the inhibitory receptor FcγRIIB has alower affinity for IgG1, IgG2 and IgG3 than all other Fcγ receptors.Bruhns et al. (2009) Blood 113:3716. Studies have shown that FcγRI doesnot bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Id. Ingeneral, with regard to ADCC activity, human IgG1≥IgG3>> IgG4≥IgG2. As aconsequence, for example, an IgG1 constant domain, rather than an IgG2or IgG4, might be chosen for use in a drug where ADCC is desired; IgG3might be chosen if activation of FcγRIIIA-expressing NK cells,monocytes, or macrophages; and IgG4 might be chosen if the antibody isto be used to desensitize allergy patients. IgG4 may also be selected ifit is desired that the antibody lack all effector function.

Accordingly, anti-OX40 variable regions described herein may be linked(e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgG1:G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); forIgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3),G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u),G3m27(v). See, e.g., Jefferis et al. (2009) mAbs 1:1). Selection ofallotype may be influenced by the potential immunogenicity concerns,e.g. to minimize the formation of anti-drug antibodies.

In certain embodiments, anti-OX40 variable regions described herein arelinked to an Fc that binds to one or more activating Fc receptors(Fc-I/CD64, FcγIIa/CD32 or FcγIIIa/CD16), and thereby stimulate ADCC andmay cause T cell depletion. In particular embodiments, anti-OX40variable regions described herein are linked to an Fc that causesdepletion. In other embodiments, anti-OX40 variable regions describedherein are linked to a human IgG1 or IgG3 Fc, i.e., the antibodies areof the IgG1 or IgG3 isotype. In other embodiments, anti-OX40 antibodiesare depleting antibodies. For example, they may deplete T_(reg) cellsthat are in the tumor microenvironment (and thereby enhance anti-tumoractivity), but not significantly deplete T_(eff) cells that are in thetumor microenvironment and mediate the anti-tumor effect, and/or notsignificantly deplete T_(reg) and T_(eff) cells that are outside of thetumor, e.g., in the periphery. In other embodiments, anti-OX40antibodies are of an isotype, (either naturally occurring ornon-naturally occurring (e.g., including mutation(s)) isotype thatstimulate T_(reg) cell depletion or elimination at the tumor site andconcomitant activation of T_(eff) cells. In other embodiments, anti-OX40antibodies create an elevated T_(eff) to T_(reg) ratio at the tumorsite, which is indicative of potent anti-tumor activity, and preferablywithout significantly depleting T_(eff) and T_(eff) cells that areoutside of the tumor, e.g., in the periphery.

In certain embodiments, anti-OX40 antibodies block the immunosuppressiveactivity of T_(regs). In other embodiments, anti-OX40 antibodies have anFc receptor with reduced or eliminated FcR binding, e.g., reducedbinding to activating FcRs. In certain embodiments, anti-OX40 antibodieshave an Fc that binds to or has enhanced binding to FcRIIb, which canprovide enhanced agonism. See, e.g., WO 2012/087928; Li & Ravetch (2011)Science 333:1030; Wilson et al. (2011) Cancer Cell 19:101; White et al.(2011) J. Immunol. 187:1754.

Anti-OX40 variable regions described herein may be linked to anon-naturally occurring Fc region, e.g., an effectorless or mostlyeffectorless Fc (e.g., human IgG2 or IgG4) or, alternatively, an Fc withenhanced binding to one or more activating Fc receptors (FcγI, FcγIIa orFcγIIIa), such as to enhance T_(reg) depletion in the tumor environment.

Variable regions described herein may be linked to an Fc comprising oneor more modification, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. Furthermore, an antibody described herein may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or it may be modified to alter its glycosylation, toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat. Sequencevariants disclosed herein are provided with reference to the residuenumber followed by the amino acid that is substituted in place of thenaturally occurring amino acid, optionally preceded by the naturallyoccurring residue at that position. Where multiple amino acids may bepresent at a given position, e.g. if sequences differ between naturallyoccurring isotypes, or if multiple mutations may be substituted at theposition, they are separated by slashes (e.g. “X/Y/Z”).

For example, one may make modifications in the Fc region in order togenerate an Fc variant with (a) increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) increased ordecreased affinity for C1q and/or (d) increased or decreased affinityfor a Fc receptor relative to the parent Fc. Such Fc region variantswill generally comprise at least one amino acid modification in the Fcregion. Combining amino acid modifications is thought to be particularlydesirable. For example, the variant Fc region may include two, three,four, five, etc substitutions therein, e.g. of the specific Fc regionpositions identified herein. Exemplary Fc sequence variants aredisclosed herein, and are also provided at U.S. Pat. Nos. 5,624,821;6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT PatentPublications WO 00/42072, WO 01/58957; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114.

Reducing Effector Function

ADCC activity may be reduced by modifying the Fc region. In certainembodiments, sites that affect binding to Fc receptors may be removed,preferably sites other than salvage receptor binding sites. In otherembodiments, an Fc region may be modified to remove an ADCC site. ADCCsites are known in the art; see, for example, Sarmay et al. (1992)Molec. Immunol. 29 (5): 633-9 with regard to ADCC sites in IgG1. In oneembodiment, the G236R and L328R variant of human IgG1 effectivelyeliminates FcγR binding. Horton et al. (2011) J. Immunol. 186:4223 andChu et al. (2008) Mol. Immunol. 45:3926. In other embodiments, the Fchaving reduced binding to FcγRs comprised the amino acid substitutionsL234A, L235E and G237A. Gross et al. (2001) Immunity 15:289.

CDC activity may also be reduced by modifying the Fc region. Mutationsat IgG1 positions D270, K322, P329 and P331, specifically alaninemutations D270A, K322A, P329A and P331A, significantly reduce theability of the corresponding antibody to bind C1q and activatecomplement. Idusogie et al. (2000) J. Immunol. 164:4178; WO 99/51642.Modification of position 331 of IgG1 (e.g. P331S) has been shown toreduce complement binding. Tao et al. (1993) J. Exp. Med. 178:661 andCanfield & Morrison (1991) J. Exp. Med. 173:1483. In another example,one or more amino acid residues within amino acid positions 231 to 239are altered to thereby reduce the ability of the antibody to fixcomplement. WO 94/29351.

In some embodiments, the Fc with reduced complement fixation has theamino acid substitutions A330S and P331S. Gross et al. (2001) Immunity15:289.

For uses where effector function is to be avoided altogether, e.g. whenantigen binding alone is sufficient to generate the desired therapeuticbenefit, and effector function only leads to (or increases the risk of)undesired side effects, IgG4 antibodies may be used, or antibodies orfragments lacking the Fc region or a substantial portion thereof can bedevised, or the Fc may be mutated to eliminate glycosylation altogether(e.g. N297A). Alternatively, a hybrid construct of human IgG2 (CH1domain and hinge region) and human IgG4 (CH2 and CH3 domains) has beengenerated that is devoid of effector function, lacking the ability tobind the FcγRs (like IgG2) and unable to activate complement (likeIgG4). Rother et al. (2007) Nat. Biotechnol. 25:1256. See also Muelleret al. (1997) Mol. Immunol. 34:441; Labrijn et al. (2008) Curr. Op.Immunol. 20:479 (discussing Fc modifications to reduce effector functiongenerally).

In other embodiments, the Fc region is altered by replacing at least oneamino acid residue with a different amino acid residue to reduce alleffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has decreased affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptor(residues 234, 235, 236, 237, 297) or the C1 component of complement(residues 297, 318, 320, 322). U.S. Pat. Nos. 5,624,821 and 5,648,260,both by Winter et al.

One early patent application proposed modifications in the IgG Fc regionto decrease binding to FcγRI to decrease ADCC (234A; 235E; 236A; G237A)or block binding to complement component C1q to eliminate CDC (E318A orV/K320A and K322A/Q). WO 88/007089. See also Duncan & Winter (1988)Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci. (USA)88:9036; and Sondermann et al. (2000) Nature 406:267 (discussing theeffects of these mutations on FcγRIII binding).

Fc modifications reducing effector function also include substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L, and328R. An Fc variant may comprise 236R/328R. Other modifications forreducing FcγR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S,229S, 238S, 233P, and 234V. These and other modifications are reviewedin Strohl (2009) Current Opinion in Biotechnology 20:685-691. Effectorfunctions (both ADCC and complement activation) can be reduced, whilemaintaining neonatal FcR binding (maintaining half-life), by mutatingIgG residues at one or more of positions 233-236 and 327-331, such asE233P, L234V, L235A, optionally G236A, A327G, A330S and P331S in IgG1;E233P, F234V, L235A, optionally G236A in IgG4; and A330S and P331S inIgG2. See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO 99/58572.Other mutations that reduce effector function include L234A and L235A inIgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A inIgG2 (Cole et al. (1997) J. Immunol. 159:3613; see also U.S. Pat. No.5,834,597); and S228P and L235E for IgG4 (Reddy et al. (2000) J.Immunol. 164:1925). Another combination of mutations for reducingeffector function in a human IgG1 include L234F, L235E and P331S.Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700.See generally Labrijn et gal. (2008) Curr. Op. Immunol. 20:479.Additional mutations found to decrease effector function in the contextof an Fc (IgG1) fusion protein (abatacept) are C226S, C229S and P238S(EU residue numbering). Davis et al. (2007) J. Immunol. 34:2204.

Other Fc variants having reduced ADCC and/or CDC are disclosed atGlaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235Ato decrease ADCC and ADCP in an IgG4); Hutchins et al. (1995) Proc.Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A and E318A in an IgG4); Anet al. (2009) MAbs 1:572 and U.S. Pat. App. Pub. 2007/0148167 (H268Q,V309L, A330S and P331S in an IgG2); McEarchern et al. (2007) Blood109:1185 (C226S, C229S, E233P, L234V, L235A in an IgG1); Vafa et al.(2014) Methods 65:114 (V234V, G237A, P238S, H268A, V309L, A330S, P331Sin an IgG2).

In certain embodiments, an Fc is chosen that has essentially no effectorfunction, i.e., it has reduced binding to FcγRs and reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless comprisesthe following five mutations: L234A, L235E, G237A, A330S and P331S.Gross et al. (2001) Immunity 15:289. Exemplary heavy chains comprisingthese mutations are set forth in the Sequence Listing, as detailed atTable 23 (e.g. SEQ ID NO: 11). These five substitutions may be combinedwith N297A to eliminate glycosylation as well.

Enhancing Effector Function

Alternatively, ADCC activity may be increased by modifying the Fcregion. With regard to ADCC activity, human IgG1≥IgG3>> IgG4≥IgG2, so anIgG1 constant domain, rather than an IgG2 or IgG4, might be chosen foruse in a drug where ADCC is desired. Alternatively, the Fc region may bemodified to increase antibody dependent cellular cytotoxicity (ADCC)and/or to increase the affinity for an Fcγ receptor by modifying one ormore amino acids at the following positions: 234, 235, 236, 238, 239,240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262,263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309,312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. See WO 2012/142515;see also WO 00/42072. Exemplary substitutions include 236A, 239D, 239E,268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T,and 267E/268F/324T. For example, human IgG1 Fcs comprising the G236Avariant, which can optionally be combined with I332E, have been shown toincrease the FcγIIA/FcγIIB binding affinity ratio approximately 15-fold.Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010)mAbs 2:181. Other modifications for enhancing FcγR and complementinteractions include but are not limited to substitutions 298A, 333A,334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L,396L, 305I, and 396L. These and other modifications are reviewed inStrohl (2009) Current Opinion in Biotechnology 20:685-691. Specifically,both ADCC and CDC may be enhanced by changes at position E333 of IgG1,e.g. E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. The use ofP247I and A339D/Q mutations to enhance effector function in an IgG1 isdisclosed in WO2006/020114, and D280H, K290S±S298DN is disclosed inWO2004/074455. The K326A/W and E333A/S variants have been shown toincrease effector function in human IgG1, and E333S in IgG2. Idusogie etal. (2001) J. Immunol. 166:2571.

Specifically, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIIIand FcRn have been mapped, and variants with improved binding have beendescribed. Shields et al. (2001) J. Biol. Chem. 276:6591-6604. Specificmutations at positions 256, 290, 298, 333, 334 and 339 were shown toimprove binding to FcγRIII, including the combination mutantsT256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A (havingenhanced FcγRIIIa binding and ADCC activity). Other IgG1 variants withstrongly enhanced binding to Fc-RIIIa have been identified, includingvariants with S239D/I332E and S239D/I332E/A330L mutations which showedthe greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIbbinding, and strong cytotoxic activity in cynomolgus monkeys. Lazar etal. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010)Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278.Introduction of the triple mutations into antibodies such as alemtuzumab(CD52-specific), trastuzumab (HER2/neu-specific), rituximab(CD20-specific), and cetuximab (EGFR-specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys. Lazar et al. (2006)Proc. Nat'l Acad Sci. (USA) 103:4005. In addition, IgG1 mutantscontaining L235V, F243L, R292P, Y300L, V305I and P396L mutations whichexhibited enhanced binding to FcγRIIIa and concomitantly enhanced ADCCactivity in transgenic mice expressing human FcγRIIIa in models of Bcell malignancies and breast cancer have been identified. Stavenhagen etal. (2007) Cancer Res. 67:8882; U.S. Pat. No. 8,652,466; Nordstrom etal. (2011) Breast Cancer Res. 13:R123.

Different IgG isotypes also exhibit differential CDC activity(IgG3>IgG1>>IgG2≈IgG4). Dangl et al. (1988) EMBO J. 7:1989. For uses inwhich enhanced CDC is desired, it is also possible to introducemutations that increase binding to C1q. The ability to recruitcomplement (CDC) may be enhanced by mutations at K326 and/or E333 in anIgG2, such as K326W (which reduces ADCC activity) and E333S, to increasebinding to C1q, the first component of the complement cascade. Idusogieet al. (2001) J. Immunol. 166:2571. Introduction of S267E/H268F/S324T(alone or in any combination) into human IgG1 enhances C1q binding.Moore et al. (2010) mAbs 2:181. The Fc region of the IgG1/IgG3 hybridisotype antibody “113F” of Natsume et al. (2008) Cancer Res. 68:3863(FIG. 1 therein) also confers enhanced CDC. See also Michaelsen et al.(2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology93:595.

Additional mutations that can increase or decrease effector function aredisclosed at Dall'Acqua et al. (2006) J. Immunol. 177:1129. See alsoCarter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol.20:460.

Fc variants that enhance affinity for the inhibitory receptor FcγRIIbmay also be used, e.g. to enhance apoptosis-inducing or adjuvantactivity. Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012)Proc. Nat'l Acad. Sci (USA) 109:10966; U.S. Pat. App. Pub. 2014/0010812.Such variants may provide an antibody with immunomodulatory activitiesrelated to FcγRllb⁺ cells, including for example B cells and monocytes.In one embodiment, the Fc variants provide selectively enhanced affinityto FcγRllb relative to one or more activating receptors. Modificationsfor altering binding to FcγRllb include one or more modifications at aposition selected from the group consisting of 234, 235, 236, 237, 239,266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.Exemplary substitutions for enhancing FcγRllb affinity include but arenot limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D,236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E,328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D,239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variantsfor enhancing binding to FcγRllb include 235Y/267E, 236D/267E,239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F. Specifically,the S267E, G236D, S239D, L328F and 1332E variants, including theS267E+L328F double variant, of human IgG1 are of particular value inspecifically enhancing affinity for the inhibitory FcγRllb receptor. Chuet al. (2008) Mol. Immunol. 45:3926; U.S. Pat. App. Pub. 2006/024298; WO2012/087928. Enhanced specificity for FcγRIIb (as distinguished fromFcγRIIa^(R131)) may be obtained by adding the P238D substitution. Mimotoet al. (2013) Protein. Eng. Des. & Selection 26:589; WO 2012/115241.

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thismay be done by increasing the binding affinity of the Fc region forFcRn. In one embodiment, the antibody is altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary Fcvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, and 434,including for example 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y,and 434M. Other variants that increase Fc binding to FcRn include: 250E,250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A,272A, 305A, 307A, 31 1A, 312A, 378Q, 380A, 382A, 434A (Shields et al,Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252Y,252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E,433K/434F/436H (Dall Acqua et al. Journal of Immunology, 2002,169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry281:23514-23524). See U.S. Pat. No. 8,367,805.

Modification of certain conserved residues in IgG Fc(I253/H310/Q311/H433/N434), such as the N434A variant (Yeung et al.(2009) J. Immunol. 182:7663), has been proposed as a way to increaseFcRn affinity, thus increasing the half-life of the antibody incirculation. WO 98/023289. The combination Fc variant comprising M428Land N434S has been shown to increase FcRn binding and increase serumhalf-life up to five-fold. Zalevsky et al. (2010) Nat. Biotechnol.28:157. The combination Fc variant comprising T307A, E380A and N434Amodifications also extends half-life of IgG1 antibodies. Petkova et al.(2006) Int. Immunol. 18:1759. In addition, combination Fc variantscomprising M252Y/M428L, M428L/N434H, M428L/N434F, M428L/N434Y,M428L/N434A, M428L/N434M, and M428L/N434S variants have also been shownto extend half-life. WO 2009/086320.

Further, a combination Fc variant comprising M252Y, S254T and T256E,increases half-life-nearly 4-fold. Dall'Acqua et al. (2006) J. Biol.Chem. 281:23514. A related IgG1 modification providing increased FcRnaffinity but reduced pH dependence (M252Y/S254T/T256E/H433K/N434F) hasbeen used to create an IgG1 construct (“MST-HN Abdeg”) for use as acompetitor to prevent binding of other antibodies to FcRn, resulting inincreased clearance of that other antibody, either endogenous IgG (e.g.in an autoimmune setting) or another exogenous (therapeutic) mAb.Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.

Other modifications for increasing FcRn binding are described in Yeunget al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO97/34631; WO 2002/060919.

In certain embodiments, hybrid IgG isotypes may be used to increase FcRnbinding, and potentially increase half-life. For example, an IgG1/IgG3hybrid variant may be constructed by substituting IgG1 positions in theCH2 and/or CH3 region with the amino acids from IgG3 at positions wherethe two isotypes differ. Thus a hybrid variant IgG antibody may beconstructed that comprises one or more substitutions, e.g., 274Q, 276K,300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In otherembodiments described herein, an IgG1/IgG2 hybrid variant may beconstructed by substituting IgG2 positions in the CH2 and/or CH3 regionwith amino acids from IgG1 at positions where the two isotypes differ.Thus a hybrid variant IgG antibody may be constructed that comprises oneor more substitutions, e.g., one or more of the following amino acidsubstitutions: 233E, 234L, 235L, −236G (referring to an insertion of aglycine at position 236), and 327A. See U.S. Pat. No. 8,629,113. Ahybrid of IgG1/IgG2/IgG4 sequences has been generated that purportedlyincreases serum half-life and improves expression. U.S. Pat. No.7,867,491 (sequence number 18 therein).

The serum half-life of the antibodies of the present invention can alsobe increased by pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with a polyethylene glycol (PEG) reagent, such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive water-soluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.In certain embodiments, the antibody to be pegylated is an aglycosylatedantibody. Methods for pegylating proteins are known in the art and canbe applied to the antibodies described herein. See for example, EP0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

Alternatively, under some circumstances it may be desirable to decreasethe half-life of an antibody, rather than increase it. Modificationssuch as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A/RI253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in Fc ofhuman IgG1 can decrease FcRn binding, thus decreasing half-life(increasing clearance) for use in situations where rapid clearance ispreferred, such a medical imaging. See also Kenanova et al. (2005)Cancer Res. 65:622. Other means to enhance clearance include formattingthe antigen binding domains of the present invention as antibodyfragments lacking the ability to bind FcRn, such as Fab fragments. Suchmodification can reduce the circulating half-life of an antibody from acouple of weeks to a matter of hours. Selective PEGylation of antibodyfragments can then be used to fine-tune (increase) the half-life of theantibody fragments if necessary. Chapman et al. (1999) Nat. Biotechnol.17:780. Antibody fragments may also be fused to human serum albumin,e.g. in a fusion protein construct, to increase half-life. Yeh et al.(1992) Proc. Nat'l Acad. Sci. 89:1904. Alternatively, a bispecificantibody may be constructed with a first antigen binding domain of thepresent invention and a second antigen binding domain that binds tohuman serum albumin (HSA). See Int'l Pat. Appl. Pub. WO 2009/127691 andpatent references cited therein. Alternatively, specialized polypeptidesequences can be added to antibody fragments to increase half-life, e.g.“XTEN” polypeptide sequences. Schellenberger et al. (2009) Nat.Biotechnol. 27:1186; Int'l Pat. Appl. Pub. WO 2010/091122.

Additional Fc Variants

When using an IgG4 constant domain, it is preferable to include thesubstitution S228P, which mimics the hinge sequence in IgG1 and therebystabilizes IgG4 molecules, e.g. reducing Fab-arm exchange between thetherapeutic antibody and endogenous IgG4 in the patient being treated.Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J.Immunol. 164:1925.

A potential protease cleavage site in the hinge of IgG1 constructs canbe eliminated by D221G and K222S modifications, increasing the stabilityof the antibody. WO 2014/043344.

The affinities and binding properties of an Fc variant for its ligands(Fc receptors) may be determined by a variety of in vitro assay methods(biochemical or immunological based assays) known in the art includingbut not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® SPR analysis), and other methods such as indirectbinding assays, competitive inhibition assays, fluorescence resonanceenergy transfer (FRET), gel electrophoresis and chromatography (e.g.,gel filtration). These and other methods may utilize a label on one ormore of the components being examined and/or employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In still other embodiments, the glycosylation of an antibody is modifiedto increase or decrease effector function. For example, an aglycoslatedantibody can be made that lacks all effector function by mutating theconserved asparagine residue at position 297 (e.g. N297A), thusabolishing complement and FcγRI binding. Bolt et al. (1993) Eur. J.Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595(using N297Q in IgG1 to eliminate glycosylation at position 297).

Although aglycosylated antibodies generally lack effector function,mutations can be introduced to restore that function. Aglycosylatedantibodies, e.g. those resulting from N297A/C/D/or H mutations orproduced in systems (e.g. E. coli) that do not glycosylate proteins, canbe further mutated to restore FcγR binding, e.g. S298G and/or T299A/G/orH (WO 2009/079242), or E382V and M428I (Jung et al. (2010) Proc. Nat'lAcad. Sci (USA) 107:604).

Additionally, an antibody with enhanced ADCC can be made by alteringglycosylation. For example, removal of fucose from heavy chainAsn297-linked oligosaccharides has been shown to enhance ADCC, based onimproved binding to FcγRIIIa. Shields et al. (2002) JBC 277:26733; Niwaet al. (2005) J. Immunol. Methods 306: 151; Cardarelli et al. (2009)Clin. Cancer Res. 15:3376 (MDX-1401); Cardarelli et al. (2010) CancerImmunol. Immunotherap. 59:257 (MDX-1342). Such low fucose antibodies maybe produced, e.g., in knockout Chinese hamster ovary (CHO) cells lackingfucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol.Bioeng. 87:614), or in other cells that generate afucosylatedantibodies. See, e.g., Zhang et al. (2011) mAbs 3:289 and Li et al.(2006) Nat. Biotechnol. 24:210 (both describing antibody production inglycoengineered Pichia pastoris.); Mossner et al. (2010) Blood 115:4393;Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa et al. (2003)J. Biol. Chem. 278:3466; EP 1176195B1. ADCC can also be enhanced asdescribed in PCT Publication WO 03/035835, which discloses use of avariant CHO cell line, Lec13, with reduced ability to attach fucose toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (see also Shields, R. L. et al.(2002) J. Biol. Chem. 277:26733-26740). Alternatively, fucose analogsmay be added to culture medium during antibody production to inhibitincorporation of fucose into the carbohydrate on the antibody (see,e.g., WO 2009/135181).

Increasing bisecting GlcNac structures in antibody-linkedoligosaccharides also enhances ADCC. PCT Publication WO 99/54342 byUmana et al. describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Additional glycosylation variants have been developed that are devoid ofgalactose, sialic acid, fucose and xylose residues (so-called GNGNglycoforms), which exhibit enhanced ADCC and ADCP but decreased CDC, aswell as others that are devoid of sialic acid, fucose and xylose(so-called G1/G2 glycoforms), which exhibit enhanced ADCC, ADCP and CDC.U.S. Pat. App. Pub. No. 2013/0149300. Antibodies having theseglycosylation patterns are optionally produced in genetically modifiedN. benthamiana plants in which the endogenous xylosyl and fucosyltransferase genes have been knocked-out.

Glycoengineering can also be used to modify the anti-inflammatoryproperties of an IgG construct by changing the α2,6 sialyl content ofthe carbohydrate chains attached at Asn297 of the Fc regions, wherein anincreased proportion of α2,6 sialylated forms results in enhancedanti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev.Immunol. 26:513. Conversely, reduction in the proportion of antibodieshaving α2,6 sialylated carbohydrates may be useful in cases whereanti-inflammatory properties are not wanted. Methods of modifying α2,6sialylation content of antibodies, for example by selective purificationof α2,6 sialylated forms or by enzymatic modification, are provided atU.S. Pat. Appl. Pub. No. 2008/0206246. In other embodiments, the aminoacid sequence of the Fc region may be modified to mimic the effect ofα2,6 sialylation, for example by inclusion of an F241A modification(see, e.g., WO 2013/095966).

VIII. Antibody Physical Properties

Antibodies described herein can contain one or more glycosylation sitesin either the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J. Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).

Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some some embodiments, the anti-OX40 antibody does notcontain variable region glycosylation. This can be achieved either byselecting antibodies that do not contain the glycosylation motif in thevariable region or by mutating residues within the glycosylation region.

In certain embodiments, the antibodies described herein do not containasparagine isomerism sites. The deamidation of asparagine may occur onN-G or D-G sequences and result in the creation of an isoaspartic acidresidue that introduces a kink into the polypeptide chain and decreasesits stability (isoaspartic acid effect). For instance, if the amino acidsequence Asp-Gly is present in the heavy and/or light chain CDRsequences of the antibody, the sequence is substituted with an aminoacid sequence that does not undergo isomerization. In one embodiment,the antibody comprises the heavy chain variable region CDR2 sequence setforth in SEQ ID NO: 76, but wherein the Asp or Gly in the Asp-Glysequence (LISYDGSRKHYADSVKG; SEQ ID NO: 76) is replaced with an aminoacid sequence that does not undergo isomerization, for example, anAsp-Ser or a Ser-Gly sequence. In another embodiment, the antibodycomprises the heavy chain variable region CDR2 sequence set forth in SEQID NO: 88, but wherein the Asp or Gly in the Asp-Gly sequence(AIDTDGGTFYADSVRG; SEQ ID NO: 88) is replaced with an amino acidsequence that does not undergo isomerization, for example, a Ser-Gly, anAsp-Ala, or a Ser-Thr sequence.

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-OX40 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, it is preferred that the T_(M1) (the temperature of initialunfolding) be greater than 60° C., preferably greater than 65° C., evenmore preferably greater than 70° C. The melting point of an antibody canbe measured using differential scanning calorimetry (Chen et al (2003)Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). Ina preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32). In certain embodiments, the anti-OX40 antibodiesdisclosed herein (e.g., the OX40.21 antibody) have increased stability.

Accordingly, in other embodiments, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

IX. Methods of Engineering Antibodies

As discussed herein, anti-OX40 antibodies having V_(H) and V_(L)sequences disclosed herein can be used to create new anti-OX40antibodies by modifying the V_(H) and/or V_(L) sequences, or theconstant region(s) of the antibodies. Thus, in another embodiment, thestructural features of anti-OX40 antibodies described herein, e.g. 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1, are used to create structurally related anti-OX40 antibodiesthat retain at least one functional property of the antibodies describedherein, such as binding to human OX40 and cynomolgus OX40. For example,one or more CDR regions of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, or mutations thereof, can becombined recombinantly with known framework regions and/or other CDRs tocreate additional, recombinantly-engineered, anti-OX40 antibodies, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, provided herein are methods for generating anti-OX40antibodies comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87, a CDR2 sequence selectedfrom the group consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76,88, and 317 and/or a CDR3 sequence selected from the group consisting ofSEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89; and (ii) a lightchain variable region antibody sequence comprising a CDR1 sequenceselected from the group consisting of SEQ ID NOs: 14, 22, 25, 34, 42,45, 54, 62, 70, 78, 81, and 90, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82,and 91, and/or a CDR3 sequence selected from the group consisting of SEQID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-OX40 antibodies described herein, which include,

-   -   (1) binding to soluble human OX40, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human OX40, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (3) binding to cynomolgus OX40, e.g., binding to membrane bound        cynomolgus OX40, e.g., with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (4) inducing or enhancing T cell activation, as evidenced by (i)        increased IL-2 and/or IFN-γ production in OX40-expressing T        cells and/or (ii) enhanced T cell proliferation;    -   (5) inhibiting the binding of OX40 ligand to OX40, e.g., with an        EC₅₀ of 1 nM or less as measured by FACS, e.g., in an assay with        hOX40-293 cells;    -   (6) binding to an epitope on the extracellular portion of mature        human OX40 (SEQ ID NO: 2), e.g., an epitope within the region        DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or        DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);    -   (7) competing for binding to human OX40 with 3F4, 14B6-1,        14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;    -   (8) competing for binding to human OX40 with 6E1-1, 6E1-2,        14A2-1, and 14A2-2.

The altered antibody may exhibit one or more, two or more, three ormore, four or more, five or more, six, or all of the functionalproperties set forth as (1) through (7) above. The functional propertiesof the altered antibodies can be assessed using standard assaysavailable in the art and/or described herein, such as those set forth inthe Examples (e.g., ELISAs, FACS).

In certain embodiments of the methods of engineering antibodiesdescribed herein, mutations can be introduced randomly or selectivelyalong all or part of an anti-OX40 antibody coding sequence and theresulting modified anti-OX40 antibodies can be screened for bindingactivity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

X. Nucleic Acid Molecules

Also provided herein are nucleic acid molecules that encode theantibodies described herein. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid is “isolated” or “rendered substantially pure”when purified away from other cellular components or other contaminants,e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g.,the chromosomal DNA that is linked to the isolated DNA in nature) orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, restriction enzymes, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. (1987) Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid can be, forexample, DNA or RNA and may or may not contain intronic sequences. In acertain embodiments, the nucleic acid is a cDNA molecule.

Nucleic acids provided herein can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules are those encoding the VH and VLsequences of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,14A2-1, 14A2-2, and 20C1. Exemplary DNA sequences encoding the VHsequences of 3F4, 14B6, 23H3, 6E1, 18E9, 8B11, 20B3, 14A2, and 20C1 areset forth in SEQ ID NOs: 126, 128, 131, 133, 136, 138, 140, 142, and145, respectively. Exemplary DNA sequences encoding the VL sequences of3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1,14A2-2, and 20C1 are set forth in SEQ ID NOs: 127, 129, 130, 132, 134,135, 137, 139, 141, 143, 144, and 146, respectively. Exemplary DNAsequences encoding heavy chain sequences are set forth in SEQ ID NOs:147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169171, 173, 175,176, and 177. Exemplary DNA sequences encoding light chain sequences areset forth in SEQ ID NOs: 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, and 174.

Exemplary nucleotide sequences encoding the mature VH and VL domains ofan anti-OX40 antibody are set forth in SEQ ID NOs: 145 and 146,respectively. An exemplary nucleotide sequence encoding the heavy chainof an anti-OX40 antibody is set forth in SEQ ID NO: 177. An exemplarynucleotide sequence encoding the light chain of an OX40 antibody is setforth in SEQ ID NO: 168.

Methods for making the anti-OX40 antibodies provided herein can includeexpressing the heavy chain and the light chains in a cell linecomprising the nucleotide sequences encoding the heavy and light chainswith a signal peptide. Host cells comprising these nucleotide sequencesare also provided herein.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, refers to the joining ofthe two DNA fragments such that the amino acid sequences they encoderemain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (hinge,CH1, CH2 and/or CH3). The sequences of human heavy chain constant regiongenes are known in the art (see e.g., Kabat, E. A., el al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1region. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VLsequences that are homologous to those of the 3F4, 14B6-1, 14B6-2, 23H3,6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 monoclonalantibodies. Exemplary nucleic acid molecules encode VH and VL sequencesthat are at least 70% identical, for example, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%identical, to nucleic acid molecules encoding the V_(H) and V_(L)sequences of the 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,20B3, 14A2-1, 14A2-2, and 20C1 monoclonal antibodies. Also providedherein are nucleic acid molecules with conservative substitutions (i.e.,substitutions that do not alter the resulting amino acid sequence upontranslation of nucleic acid molecule), e.g., for codon optimization.

XI. Antibody Generation

Anti-OX40 antibodies described herein can be produced using a variety ofknown techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein, Nature 256: 495 (1975).Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies also canbe employed, e.g., viral or oncogenic transformation of B lymphocytes,phage display technique using libraries of human antibody genes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies described herein can be prepared basedon the sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In one embodiment, the antibodies described herein are human monoclonalantibodies. Such human monoclonal antibodies directed against OX40 canbe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In certain embodiments, antibodies described herein are raised using amouse that carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-OX40 antibodies described herein. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-OX40 antibodies described herein. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-OX40antibodies described herein.

Additional mouse systems described in the art for raising humanantibodies, e.g., human anti-OX40 antibodies, include (i) theVelocImmune® mouse (Regeneron Pharmaceuticals, Inc.), in which theendogenous mouse heavy and light chain variable regions have beenreplaced, via homologous recombination, with human heavy and light chainvariable regions, operatively linked to the endogenous mouse constantregions, such that chimeric antibodies (human V/mouse C) are raised inthe mice, and then subsequently converted to fully human antibodiesusing standard recombinant DNA techniques; and (ii) the MeMo® mouse(Merus Biopharmaceuticals, Inc.), in which the mouse containsunrearranged human heavy chain variable regions but a single rearrangedhuman common light chain variable region. Such mice, and use thereof toraise antibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies described herein can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunizations

To generate fully human antibodies to OX40, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the OX40 antigen and/or cells expressing OX40 or fragmentthereof, as described for other antigens, for example, by Lonberg et al.(1994) Nature 368(6474): 856-859; Fishwild et al. (1996) NatureBiotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can beimmunized with DNA encoding human OX40 or fragment thereof. Preferably,the mice will be 6-16 weeks of age upon the first infusion. For example,a purified or enriched preparation (5-50 μg) of the recombinant OX40antigen can be used to immunize the HuMAb mice intraperitoneally. In theevent that immunizations using a purified or enriched preparation of theOX40 antigen do not result in antibodies, mice can also be immunizedwith cells expressing OX40, e.g., a cell line, to promote immuneresponses. Exemplary cell lines include OX40-overexpressing stable CHOand Raji cell lines.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in Ribi's adjuvant, followed byevery other week IP/SC immunizations (up to a total of 10) with antigenin Ribi's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and FACS (asdescribed below), and mice with sufficient titers of anti-OX40 humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen and lymph nodes. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually, HCo7, HCo12, and KMstrains are used. In addition, both HCo7 and HCo12 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12).

Generation of Hybridomas Producing Monoclonal Antibodies to OX40

To generate hybridomas producing the antibodies described herein,splenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSp2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.Cells are plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 10%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1× HAT (Sigma). After approximately two weeks,cells can be cultured in medium in which the HAT is replaced with HT.Individual wells can then be screened by ELISA for human monoclonal IgMand IgG antibodies. Once extensive hybridoma growth occurs, medium canbe observed usually after 10-14 days. The antibody secreting hybridomascan be replated, screened again, and if still positive for human IgG,the monoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify the antibodies, selected hybridomas can be grown in two-literspinner-flasks for monoclonal antibody purification. Supernatants can befiltered and concentrated before affinity chromatography with proteinA-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked bygel electrophoresis and high performance liquid chromatography to ensurepurity. The buffer solution can be exchanged into PBS, and theconcentration can be determined by OD280 using 1.43 extinctioncoefficient. The antibodies can then be aliquoted and stored at −80° C.

Generation of Transfectoma Producing Monoclonal Antibodies to OX40

Antibodies can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (Morrison, S. (1985)Science 229:1202).

For example, to express antibodies, or antibody fragments thereof, DNAsencoding partial or full-length light and heavy chains, can be obtainedby standard molecular biology techniques (e.g., PCR amplification orcDNA cloning using a hybridoma that expresses the antibody of interest)and the DNAs can be inserted into expression vectors such that the genesare operatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or both genes are insertedinto the same expression vector. The antibody genes are inserted intothe expression vector(s) by standard methods (e.g., ligation ofcomplementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the CH segment(s)within the vector and the V_(L) segment is operatively linked to the CLsegment within the vector.

Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein). Exemplary signal sequences for usein antibody heavy and light chains include the signal sequencesoriginally found in the anti-OX40 antibodies described herein.

In addition to the antibody chain genes, recombinant expression vectorsmay carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may beused, such as the ubiquitin promoter or β-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SRα promoter system, which contains sequences fromthe SV40 early promoter and the long terminal repeat of human T cellleukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences,recombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr− host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies described herein in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13). The antibodies disclosed herein canalso be produced in glycoengineered strains of the yeast Pichiapastoris. Li et al. (2006) Nat. Biotechnol. 24:210.

Preferred mammalian host cells for expressing the recombinant antibodiesdescribed herein include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

The N- and C-termini of antibody polypeptide chains of the presentinvention may differ from the expected sequence due to commonly observedpost-translational modifications. For example, C-terminal lysineresidues are often missing from antibody heavy chains. Dick et al.(2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues, andto a lesser extent glutamate residues, are frequently converted topyroglutamate residues on both light and heavy chains of therapeuticantibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al.(2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211.

XII. Assays

Anti-OX40 antibodies described herein can be tested for binding to OX40by, for example, standard ELISA. Briefly, microtiter plates are coatedwith purified OX40 at 1-2 μg/ml in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom OX40-immunized mice) are added to each well and incubated for 1-2hours at 37° C. The plates are washed with PBS/Tween and then incubatedwith secondary reagent (e.g., for human antibodies, a goat-anti-humanIgG Fc-specific polyclonal reagent) conjugated to horseradish peroxidase(HRP) for 1 hour at 37° C. After washing, the plates are developed withABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by aspectrophotometer at OD 415-495. Sera from immunized mice are thenfurther screened by flow cytometry for binding to a cell line expressinghuman OX40, but not to a control cell line that does not express OX40.Briefly, the binding of anti-OX40 antibodies is assessed by incubatingOX40 expressing CHO cells with the anti-OX40 antibody at 1:20 dilution.The cells are washed and binding is detected with a PE-labeledanti-human IgG Ab. Flow cytometric analyses are performed using aFACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Preferably,mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the OX40 immunogen. Hybridomas that produce antibodiesthat bind, preferably with high affinity, to OX40 can then be subclonedand further characterized. One clone from each hybridoma, which retainsthe reactivity of the parent cells (by ELISA), can then be chosen formaking a cell bank, and for antibody purification.

To purify anti-OX40 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-OX40 antibodies bind to uniqueepitopes, each antibody can be biotinylated using commercially availablereagents (Pierce, Rockford, Ill.). Biotinylated MAb binding can bedetected with a streptavidin labeled probe. Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using OX40 coated-ELISA plates as described above.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To test the binding of antibodies to live cells expressing OX40, flowcytometry can be used, as described in the Examples. Briefly, cell linesexpressing membrane-bound OX40 (grown under standard growth conditions)are mixed with various concentrations of monoclonal antibodies in PBScontaining 0.1% BSA at 4° C. for 1 hour. After washing, the cells arereacted with Fluorescein-labeled anti-IgG antibody under the sameconditions as the primary antibody staining. The samples can be analyzedby FACScan instrument using light and side scatter properties to gate onsingle cells and binding of the labeled antibodies is determined. Analternative assay using fluorescence microscopy may be used (in additionto or instead of) the flow cytometry assay. Cells can be stained exactlyas described above and examined by fluorescence microscopy. This methodallows visualization of individual cells, but may have diminishedsensitivity depending on the density of the antigen.

Anti-OX40 antibodies can be further tested for reactivity with the OX40antigen by Western blotting. Briefly, cell extracts from cellsexpressing OX40 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics include standard assays known in the art, for example, BIACORE®SPR analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala,Sweden).

In certain embodiments, the anti-OX40 antibody specifically binds to theextracellular region of human OX40. For example, the antibody mayspecifically bind to a particular domain (e.g., a functional domain)within the extracellular domain of OX40. In a particular embodiment, theantibody specifically binds to the site on OX40 to which OX40-L binds.In other embodiments, the antibody specifically binds to theextracellular region of human OX40 and the extracellular region ofcynomolgus OX40.

XIII. Immunoconjugates, Antibody Derivatives and Diagnostics

Anti-OX40 antibodies described herein can be used for diagnosticpurposes, including sample testing and in vivo imaging, and for thispurpose the antibody (or binding fragment thereof) can be conjugated toan appropriate detectable agent, to form an immunoconjugate. Fordiagnostic purposes, appropriate agents are detectable labels thatinclude radioisotopes, for whole body imaging, and radioisotopes,enzymes, fluorescent labels and other suitable antibody tags for sampletesting.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labelsincluding metal sols such as colloidal gold, isotopes such as I¹²⁵ orTc⁹⁹ presented for instance with a peptidic chelating agent of the N₂S₂,N₃S or N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated antibody would then bedetectable by avidin or streptavidin binding. Suitable enzyme labelsinclude horseradish peroxidase, alkaline phosphatase and the like. Forinstance, the label can be the enzyme alkaline phosphatase, detected bymeasuring the presence or formation of chemiluminescence followingconversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-Star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

Preferably, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-),sulfide-, (sterically hindered), disulfide-, hydrazone-, and etherlinkages. These linkages are nearly non-immunogenic and show reasonablestability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol.13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibody,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant polypeptide of between 50 to 500amino acids, there are standard procedures in text books describing thechemistry for synthesis of protein conjugates, which can be easilyfollowed by the skilled artisan (see e.g. Hackenberger, C. P. R., andSchwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). Inone embodiment the reaction of a maleinimido moiety with a cysteineresidue within the antibody or the moiety is used. This is an especiallysuited coupling chemistry in case e.g. a Fab or Fab′-fragment of anantibody is used. Alternatively in one embodiment coupling to theC-terminal end of the antibody or moiety is performed. C-terminalmodification of a protein, e.g. of a Fab-fragment can e.g. be performedas described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009)3361-3371).

In general, site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C—N bonds is described in Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403).

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents.

The reactivity of an N-terminal cysteine with benzonitrils (see Ren etal., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cysteine residues(Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology(2009), 22 (Protein Engineering), 65-96).

EP 1 074 563 describes a conjugation method which is based on the fasterreaction of a cysteine within a stretch of negatively charged aminoacids than a cysteine located in a stretch of positively charged aminoacids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a greatvariety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide, the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non-labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

In one embodiment, the moiety attached to the anti-OX40 antibody isselected from the group consisting of a binding moiety, a labelingmoiety, and a biologically active moiety.

Anti-OX40 antibodies described herein also may be conjugated to atherapeutic agent to form an immunoconjugate such as an antibody-drugconjugate (ADC). Suitable therapeutic agents include antimetabolites,alkylating agents, DNA minor groove binders, DNA intercalators, DNAcrosslinkers, histone deacetylase inhibitors, nuclear export inhibitors,proteasome inhibitors, topoisomerase I or II inhibitors, heat shockprotein inhibitors, tyrosine kinase inhibitors, antibiotics, andanti-mitotic agents. In the ADC, the antibody and therapeutic agentpreferably are conjugated via a linker cleavable such as a peptidyl,disulfide, or hydrazone linker. More preferably, the linker is apeptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys,Pro-Val-Gly-Val-Val (SEQ ID NO: 180), Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADCs can beprepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and7,129,261; PCT Publications WO 02/096910; WO 07/038658; WO 07/051081; WO07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publications20060024317; 20060004081; and 20060247295; the disclosures of which areincorporated herein by reference.

More specifically, in an ADC, the antibody is conjugated to a drug, withthe antibody functioning as a targeting agent for directing the ADC to atarget cell expressing its antigen, such as a cancer cell. Preferably,the antigen is a tumor associated antigen, i.e., one that is uniquelyexpressed or overexpressed by the cancer cell. Once there, the drug isreleased, either inside the target cell or in its vicinity, to act as atherapeutic agent. For a review on the mechanism of action and use ofADCs in cancer therapy, see Schrama et al., Nature Rev. Drug Disc. 2006,5, 147.

For cancer treatment, the drug preferably is a cytotoxic drug thatcauses death of the targeted cancer cell. Cytotoxic drugs that can beused in ADCs include the following types of compounds and their analogsand derivatives:

-   (a) enediynes such as calicheamicin (see, e.g., Lee et al., J. Am.    Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see, e.g.,    Davies et al., WO 2007/038868 A2 (2007) and Chowdari et al., U.S.    Pat. No. 8,709,431 B2 (2012));-   (b) tubulysins (see, e.g., Domling et al., U.S. Pat. No. 7,778,814    B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922 B2 (2013); and Cong    et al., U.S. Ser. No. 14/177,376, filed Feb. 11, 2014));-   (c) CC-1065 and duocarmycin (see, e.g., Boger, U.S. Pat. No.    6,5458,530 B1 (2003); Sufi et al., U.S. Pat. No. 8,461,117 B2    (2013); and Zhang et al., US 2012/0301490 A1 (2012));-   (d) epothilones (see, e.g., Vite et al., US 2007/0275904 A1 (2007)    and U.S. RE42930 E (2011));-   (e) auristatins (see, e.g., Senter et al., U.S. Pat. No. 6,844,869    B2 (2005) and Doronina et al., U.S. Pat. No. 7,498,298 B2 (2009));-   (f) pyrrolobezodiazepine (PBD) dimers (see, e.g., Howard et al., US    2013/0059800 A1(2013); US 2013/0028919 A1 (2013); and WO 2013/041606    A1 (2013)); and-   (g) maytansinoids such as DM1 and DM4 (see, e.g., Chari et al., U.S.    Pat. No. 5,208,020 (1993) and Amphlett et al., U.S. Pat. No.    7,374,762 B2 (2008)).

In an ADC, a linker covalently connects the antibody and the drug.Typically, there is one drug molecule attached to each linker, but thelinker can be branched, allowing the attachment of plural drug moleculesto increase the drug payload delivered per ADC. Further, each antibodymay have more than one linker attached. The number drug moleculescarried on an ADC is referred to as the drug-antibody ratio (DAR). Forinstance, if each heavy chain of the antibody has attached to it onelinker that in turn has one drug molecule attached, the DAR is 2.Preferably, the DAR is between 1 and 5, more preferably between 2 and 4.Those skilled in the art will also appreciate that, while in eachindividual ADC the antibody is conjugated to an integer number of drugmolecules, as a whole, a preparation of the ADC may analyze for anon-integer DAR, reflecting a statistical average. In summary, thearchitecture of an ADC may be represented by the following formula:

[Antibody]-[Linker-(Drug)n]m

where typically m is 1, 2, 3, 4, 5, or 6 (preferably 2, 3, or 4) and nis 1, 2, or 3.

In some embodiments, the linker contains a cleavable group that iscleaved inside or in the vicinity of the target cell, to release thedrug. In other embodiments, the linker does not contain a cleavablegroup but, rather, the ADC relies on catabolism of the antibody torelease the drug.

One type of cleavable group is a pH sensitive group. The pH in bloodplasma is slightly above neutral, while the pH inside a lysosome—wheremost ADCs end up after internalization inside a target cell—is acidic,circa 5. Thus, a cleavable group whose cleavage is acid catalyzed willcleave at a rate several orders of magnitude faster inside a lysosomethan in the blood plasma. Examples of acid-sensitive groups includecis-aconityl amides and hydrazones, as described in Shen et al., U.S.Pat. No. 4,631,190 (1986); Shen et al., U.S. Pat. No. 5,144,011 (1992);Shen et al., Biochem. Biophys. Res. Commun. 1981, 102, 1048; and Yang etal., Proc. Nat'l Acad. Sci (USA), 1988, 85, 1189; the disclosures ofwhich are incorporated herein by reference.

In another embodiment, the cleavable group is a disulfide. Disulfidescan be cleaved by a thiol-disulfide exchange mechanism, at a ratedependent on the ambient thiol concentration. As the intracellularconcentration of glutathione and other thiols is higher than their serumconcentrations, the cleavage rate of a disulfide will be higherintracellularly, i.e., after internalization of the ADC. Further, therate of thiol-disulfide exchange can be modulated by adjustment of thesteric and electronic characteristics of the disulfide (e.g., analkyl-aryl disulfide versus an alkyl-alkyl disulfide; substitution onthe aryl ring, etc.), enabling the design of disulfide linkages thathave enhanced serum stability or a particular cleavage rate. Foradditional disclosures relating to disulfide cleavable groups inconjugates, see, e.g., Thorpe et al., Cancer Res. 1988, 48, 6396; Santiet al., U.S. Pat. No. 7,541,530 B2 (2009); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1 (2002); Boyd et al.,U.S. Pat. No. 7,691,962 B2 (2010); and Sufi et al., US 2010/0145036 A1(2010); the disclosures of which are incorporated herein by reference.

A preferred cleavable group is a peptide that is cleaved selectively bya protease inside the target cell, as opposed to by a protease in theserum. Typically, a cleavable peptide group comprises from 1 to 20 aminoacids, preferably from 1 to 6 amino acids, more preferably from 1 to 3amino acids. The amino acid(s) can be natural and/or non-natural a-aminoacids. Natural amino acids are those encoded by the genetic code, aswell as amino acids derived therefrom, e.g., hydroxyproline,7-carboxyglutamate, citrulline, and O-phosphoserine. In this context,the term “amino acid” also includes amino acid analogs and mimetics.Analogs are compounds having the same general H₂N(R)CHCO₂H structure ofa natural amino acid, except that the R group is not one found among thenatural amino acids. Examples of analogs include homoserine, norleucine,methionine-sulfoxide, and methionine methyl sulfonium. An amino acidmimetic is a compound that has a structure different from the generalchemical structure of an a-amino acid but functions in a manner similarto one. The amino acid can be of the “L” stereochemistry of thegenetically encoded amino acids, as well as of the enantiomeric “D”stereochemistry.

Preferably, a cleavable peptide group contains an amino acid sequencethat is a cleavage recognition sequence for a protease. Many cleavagerecognition sequences are known in the art. See, e.g., Matayoshi et al.Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994);Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol.244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith etal. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol.248: 614 (1995); the disclosures of which are incorporated herein byreference.

More preferably, a cleavable peptide group comprises an amino acidsequence selected for cleavage by an endosomal or lysosomal protease,especially the latter. Examples of such proteases include cathepsins B,C, D, H, L and S, especially cathepsin B. Cathepsin B preferentiallycleaves peptides at a sequence -AA²-AA¹- where AA¹ is a basic orstrongly hydrogen bonding amino acid (such as lysine, arginine, orcitrulline) and AA² is a hydrophobic amino acid (such as phenylalanine,valine, alanine, leucine, or isoleucine), for example Val-Cit (where Citdenotes citrulline) or Val-Lys, written in the N-to-C direction. Foradditional information regarding cathepsin-cleavable groups, seeDubowchik et al., Biorg. Med. Chem. Lett. 1998, 8, 3341; Dubowchik etal., Bioorg. Med. Chem. Lett., 1998, 8, 3347; and Dubowchik et al.,Bioconjugate Chem. 2002, 13, 855; the disclosures of which areincorporated by reference. Another enzyme that can be utilized forcleaving peptidyl linkers is legumain, a lysosomal cysteine proteasethat preferentially cleaves at Ala-Ala-Asn.

In a preferred embodiment, the linker in ADCs comprises a di- ortripeptide that is preferentially cleaved by a protease located insidethe target cell. Preferably, the di- or tripeptide is cleavable bycathepsin B, more preferably a Val-Cit or Val-Lys dipeptide.

Single amino acid cleavable peptide groups also can be used, asdisclosed in Chen et al., US 2010/0113476 A1 (2010), the disclosure ofwhich is incorporated herein by reference.

For conjugates that are not intended to be internalized by a cell, thecleavable group can be chosen such that it is cleaved by a proteasepresent in the extracellular matrix in the vicinity of the target cell,e.g., a protease released by nearby dying cells or a tumor-associatedprotease. Exemplary extracellular tumor-associated proteases are matrixmetalloproteases (MMP), plasmin, thimet oligopeptidase (TOP) and CD10.See, e.g., Trouet et al., U.S. Pat. No. 5,962,216 (1999) and U.S. Pat.No. 7,402,556 B2 (2008); Dubois et al., U.S. Pat. No. 7,425,541 B2(2008); and Bebbington et al., U.S. Pat. No. 6,897,034 B2 (2005); thedisclosures of which are incorporated herein by reference.

The linker can perform other functions in addition to covalently linkingthe antibody and the drug. For instance, the linker can containpoly(ethylene glycol) (PEG) groups, which enhance solubility eitherduring the performance the conjugation chemistry or in the final ADCproduct.

The linker can further include a self-immolating moiety located adjacentto a cleavable peptide group. The self-immolating group serves as aspacer that prevents the antibody and/or the drug moiety from stericallyinterfering with the cleavage of the peptide group by a protease butthereafter spontaneously releases itself (i.e., self-immolates) so as tonot interfere with the action of the drug. See Carl et al., J. Med.Chem. 1981, 24 (3), 479; Carl et al., WO 81/01145 (1981); Dubowchik etal., Pharmacology & Therapeutics 1999, 83, 67; Firestone et al., U.S.Pat. No. 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 2002, 67, 1866;Doronina et al., Nature Biotechnology 2003, 21 (7), 778 (erratum, p.941); de Groot et al., Org. Chem. 2001, 66, 8815; Boyd et al., U.S. Pat.No. 7,691,962 B2 (2010); Boyd et al., US 2008/0279868 A1 (2008); Sufi etal., WO 2008/083312 A2 (2008); Feng, U.S. Pat. No. 7,375,078 B2 (2008);Jeffrey, U.S. Pat. No. 8,039,273 B2 (2011); and Senter et al., US2003/0096743 A1 (2003); the disclosures of which are incorporated byreference.

A preferred self-immolating group is a p-aminobenzyl oxycarbonyl (PABC)group, whose structure and mechanism of action is depicted below:

Thus, in a preferred embodiment, an ADC has a linker comprising a di- ortripeptide that is preferentially cleaved by a protease located insidethe target cell and, adjacent to the di- or tripeptide, aself-immolating group. Preferably, the di- or tripeptide is cleavable bycathepsin B. Preferably, the self-immolating group is a PABC group.

Numerous techniques can be used for conjugating the antibody and thedrug. In a preferred one, an E-amino group in the side chain of a lysineresidue in the antibody is reacted with 2-iminothiolane to introduce afree thiol (—SH) group. The thiol group can react with a maleimide orother nucleophile acceptor group to effect conjugation, as illustratedbelow:

Typically, a thiolation level of two to three thiols per antibody isachieved. For a representative procedure, see Chowdari et al., U.S. Pat.No. 8,709,431 B2 (2014), the disclosure of which is incorporated hereinby reference. Thus, in one embodiment, an antibody of this invention hasone or more lysine residues (preferably two or three) modified byreaction with iminothiolane.

An alternative conjugation technique employs copper-free “clickchemistry,” in which an azide group adds across the strained alkyne bondof a cyclooctyne to form an 1,2,3-triazole ring. See, e.g., Agard etal., J. Amer. Chem. Soc. 2004, 126, 15046; Best, Biochemistry 2009, 48,6571, the disclosures of which are incorporated herein by reference. Theazide can be located on the antibody and the cyclooctyne on the drugmoiety, or vice-versa. A preferred cyclooctyne group isdibenzocyclooctyne (DIBO). Various reagents having a DIBO group areavailable from Invitrogen/Molecular Probes, Eugene, Oreg. The reactionbelow illustrates click chemistry conjugation for the instance in whichthe DIBO group is attached to the antibody:

In an ADC made by this technique, the linker comprises a 1,2,3-triazolering.

Yet another conjugation technique involves introducing a non-naturalamino acid into an antibody, with the non-natural amino acid providing afunctionality for conjugation with a reactive functional group in thedrug moiety. For instance, the non-natural amino acidp-acetylphenylalanine can be incorporated into an antibody or otherpolypeptide, as taught in Tian et al., WO 2008/030612 A2 (2008). Theketone group in p-acetylphenyalanine can be a conjugation site by theformation of an oxime with a hydroxylamino group on the linker-drugmoiety. Alternatively, the non-natural amino acid p-azidophenylalaninecan be incorporated into an antibody to provide an azide functionalgroup for conjugation via click chemistry, as discussed above.Non-natural amino acids can also be incorporated into an antibody orother polypeptide using cell-free methods, as taught in Goerke et al.,US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009,102 (2), 400-416. The foregoing disclosures are incorporated herein byreference. Thus, in one embodiment, the antibody has one or more aminoacids replaced by a non-natural amino acid, which preferably isp-acetylphenylalanine or p-azidophenylalanine, more preferablyp-acetylphenylalanine.

Still another conjugation technique uses the enzyme transglutaminase(preferably bacterial transglutaminase or BTG), as taught in Jeger etal., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG forms an amide bondbetween the side chain carboxamide of a glutamine and an alkyleneaminogroup, which can be, for example, the E-amino group of a lysine or a5-amino-n-pentyl group. In a typical conjugation reaction, the glutamineresidue is located on the antibody, while the alkyleneamino group islocated on the linker-drug moiety, as shown below:

The positioning of a glutamine residue on a polypeptide chain has alarge effect on its susceptibility to BTG mediated transamidation. Noneof the glutamine residues on an antibody are normally BTG substrates.However, if the antibody is deglycosylated—the glycosylation site beingasparagine 297 (N297)—nearby glutamine 295 (Q295) is rendered BTGsusceptible. Alternatively, an antibody can be synthesized glycosidefree by introducing an N297A mutation in the constant region, toeliminate the N297 glycosylation site. Further, it has been shown thatan N297Q substitution in an antibody not only eliminates glycosylation,but also introduces a second glutamine residue (at position 297) thattoo is susceptible BTG-mediated transamidation. Thus, in one embodiment,the anti-OX40 antibody is deglycosylated. In another embodiment, theanti-OX40 antibody has an N297Q substitution. Those skilled in the artwill appreciate that deglycosylation by post-synthesis modification orby introducing an N297A mutation generates two BTG-reactive glutamineresidues per antibody (one per heavy chain, at position 295), while anantibody with an N297Q substitution will have four BTG-reactiveglutamine residues (two per heavy chain, at positions 295 and 297).

Further, another conjugation technique uses the enzyme Sortase A, astaught in Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol.Lett. 2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Maoet al., WO 2005/051976 A2 (2005), the disclosures of which areincorporated herein by reference. The Sortase A recognition motif(typically LPXTG (SEQ ID NO: 181), where X is any natural amino acid)may be attached to the antibody and the nucleophilic acceptor motif(typically GGG) may be located on the drug moiety, or vice-versa.

Anti-OX40 antibodies described herein also can be used for detectingOX40, such as human OX40, e.g., human OX40 in tissues or tissue samples.The antibodies may be used, e.g., in an ELISA assay or in flowcytometry. In certain embodiments, the anti-OX40 antibody is contactedwith cells, e.g., cells in a tissue, for a time appropriate for specificbinding to occur, and then a reagent, e.g., an antibody that detects theanti-OX40 antibody, is added. Exemplary assays are provided in theExamples. The anti-OX40 antibody may be a fully human antibody, or itmay be a chimeric antibody, such as an antibody having human variableregions and murine constant regions or a portion thereof. Exemplarymethods for detecting OX40, e.g., human OX40, in a sample (cell ortissue sample) comprise (i) contacting a sample with an anti-OX40antibody, for a time sufficient for allowing specific binding of theanti-OX40 antibody to OX40 in the sample, and (2) contacting the samplewith a detection reagent, e.g., an antibody, that specifically binds tothe anti-OX40 antibody, such as to the Fc region of the anti-OX40antibody, to thereby detect OX40 bound by the anti-OX40 antibody. Washsteps may be included after the incubation with the antibody and/ordetection reagent. Anti-OX40 antibodies for use in these methods do nothave to be linked to a label or detection agents, as a separatedetection agent can be used.

XIV. Bispecific Molecules

Anti-OX40 antibodies described herein may be used for forming bispecificmolecules. For example, the antibody, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. In one embodiment, theanti-OX40 antibody may be linked to an antibody or scFv that bindsspecifically to any protein that may be used as potential targets forcombination treatments, such as the proteins described herein (e.g.,antibodies to PD-1, PD-L1, or LAG-3). Alternatively, the antibody may bederivatized or linked to more than one other functional molecule togenerate multispecific molecules that bind to more than two differentbinding sites and/or target molecules. Such multispecific molecules arealso intended to be encompassed by the term “bispecific molecule” asused herein. To create a bispecific molecule described herein, theanti-OX40 antibody can be functionally linked (e.g., by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other binding molecules, such as another antibody, antibodyfragment, peptide or binding mimetic, such that a bispecific moleculeresults.

Accordingly, provided herein are bispecific molecules comprising atleast one first binding specificity for OX40 and a second bindingspecificity for a second target epitope. In one embodiment, thebispecific molecule is multispecific, e.g., the molecule furtherincludes a third binding specificity.

In certain embodiments, the bispecific molecules comprises as a bindingspecificity at least one antibody, or an antibody fragment thereof,including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv (scFv).The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies can beemployed in the bispecific molecules described herein, including, e.g.,murine, chimeric and humanized antibodies.

Bispecific molecules provided herein can be prepared by conjugating theconstituent binding specificities using methods known in the art. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,mAb×(scFv)₂, Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecificantibody may comprise an antibody comprising an scFv at the C-terminusof each heavy chain. A bispecific molecule described herein can be asingle chain molecule comprising one single chain antibody and a bindingdeterminant, or a single chain bispecific molecule comprising twobinding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed using art-recognized methods, such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,bioassay (e.g., growth inhibition), or Western Blot assay. Each of theseassays generally detects the presence of protein-antibody complexes ofparticular interest by employing a labeled reagent (e.g., an antibody)specific for the complex of interest.

XV. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions,containing one or more anti-OX40 antibodies, alone or in combinationwith antibodies to other targets, formulated together with apharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) antibodies, orimmunoconjugates or bispecific molecules described herein. For example,the composition can comprise a combination of antibodies (orimmunoconjugates or bispecifics) described herein that bind to differentepitopes on OX40 or that have complementary activities.

In certain embodiments, the composition comprises an anti-OX40 antibodyat a concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 1-300 mg/ml, or 100-300 mg/ml.

Pharmaceutical compositions described herein also can be administered incombination therapies, i.e., combined with other agents. For example,the combination therapy can include administration of an anti-OX40antibody described herein combined with at least one other anti-cancerand/or T-cell stimulating (e.g., activating) agent. Examples oftherapeutic agents that can be used in combination therapy are describedin greater detail below in the section on uses of the antibodiesdescribed herein.

In certain embodiments, therapeutic compositions disclosed hereininclude other compounds, drugs, and/or agents used for the treatment ofcancer. Such compounds, drugs, and/or agents can include, for example,chemotherapy drugs, small molecule drugs or antibodies that stimulatethe immune response to a given cancer. In some instances, therapeuticcompositions can include, for example, one or more of an anti-CTLA-4antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-GITRantibody, an anti-CD137 antibody, or an anti-LAG-3 antibody.

As used herein, “pharmaceutically acceptable carriers” include any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compositions described herein may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical compositions described herein also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. A pharmaceutical composition maycomprise a preservative or may be devoid of a preservative.Supplementary active compounds can be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the anti-OX40 antibody, the dosage ranges fromabout 0.0001 to 100 mg/kg, about 0.01 to 5 mg/kg, about 0.01 to 10mg/kg, about 0.1 to 1 mg/kg, about 0.1 to 0.5 mg/kg, or about 0.5 to 0.8mg/kg of the host body weight. For example, dosages can be 0.2 mg/kgbody weight, 0.3 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. In certain embodiments, the dosage is0.2 mg/kg. In some embodiments, the dosage is 0.25 mg/kg. In otherembodiments, the dosage is 0.5 mg/kg. An exemplary treatment regimeentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months. Exemplary dosage regimens for theantibodies described herein include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

In certain embodiments, for combination treatment with an anti-OX40antibody and anti-PD-1 or anti-CTLA-4 antibody, the antibodies areadministered at a fixed dose. Accordingly, in some embodiments, theanti-OX40 antibody is administered at a fixed dose of about 25 to about320 mg, for example, about 25 to about 160 mg, about 25 to about 80 mg,about 25 to about 40 mg, about 40 to about 320 mg, about 40 to about 160mg, about 40 to about 80 mg, about 80 to about 320 mg, about 30 to about160 mg, or about 160 to about 320 mg. In one embodiment, the anti-OX40antibody is administered at a dose of 20 mg or about 20 mg. In anotherembodiment, the anti-OX40 antibody is administered at a dose of 40 mg orabout 40 mg. In another embodiment, the anti-OX40 antibody isadministered at a dose of 80 mg or about 80 mg. In another embodiment,the anti-OX40 antibody is administered at a dose of 160 mg or about 160mg. In another embodiment, the anti-OX40 antibody is administered at adose of 320 mg or about 320 mg.

In some embodiments, the anti-PD-1 antibody is administered at a fixeddose of about 100 to 300 mg, For example, the dosage of theimmuno-oncology agent can be 240 mg or about 240 mg, 360 mg or about 360mg, or 480 mg or about 480 mg. In certain embodiments, the dose of theanti-PD1 antibody ranges from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg, of the host body weight. For example dosagescan be 0.3 mg/kg body weight or about 0.3 mg/kg body weight, 1 mg/kgbody weight or about 1 mg/kg body weight, 3 mg/kg body weight or about 3mg/kg body weight, 5 mg/kg body weight or about 5 mg/kg body weight, or10 mg/kg body weight or about 10 mg/kg body weight, or within the rangeof 1-10 mg/kg. In some embodiments, the dosage of the anti-PD-1 antibodyis 240 mg or about 240 mg administered once every 2 weeks (Q2W). Thisdosage can be adjusted proportionately (at 120 mg per week) for longeror shorter periods, e.g., 360 mg administered once every 3 weeks (Q3W)or 480 mg administered once every 4 weeks (Q4W).

In some embodiments, the anti-CTLA-4 antibody is administered at a doseof about 0.1 mg/kg to about 10 mg/kg. For example, dosages can be 1mg/kg or about 1 mg/kg or 3 mg/kg or about 3 mg/kg, of the host bodyweight.

Exemplary dosage regimens for combination treatment with an anti-OX40and anti-PD-1 or anti-CTLA-4 antibody are provided infra under thesection titled “Uses and Methods.”

In certain embodiments, the anti-OX40 antibody is administered to apatient with an infusion duration of about 15 minutes to about 60minutes, for example, about 30 minutes.

In certain embodiments, the anti-PD-1 antibody (e.g., nivolumab) isadministered to a patient with an infusion duration of about 15 minutesto about 60 minutes, for example, about 30 minutes, when administered ata dose of 3 mg/kg (0.1 mg/kg/min). In certain embodiments, the anti-PD-1antibody is administered to a patient with an infusion duration of about45 minutes to 75 minutes, for example, about 60 minutes, whenadministered at a dose of 10 mg/kg.

In certain embodiments, the anti-CTLA-4 antibody (e.g., ipilimumab) isadministered to a patient with an infusion duration of about 15 minutesto 120 minutes, for example, about 30 minutes when administered at adose of 3 mg/kg. In certain embodiments, the anti-CTLA-4 antibody isadministered to a patient with an infusion duration of about 15 minutesto 120 minutes, for example, 90 minutes, when administered at a dose of10 mg/kg.

In certain embodiments, when administered on the same day, the anti-OX40antibody is administered before the anti-PD-1 or anti-CTLA-4 antibody.In certain embodiments, when administered on the same day, the anti-OX40antibody is administered after the anti-PD-1 or anti-CTLA-4 antibody. Incertain embodiments, when administered on the same day, the anti-OX40antibody is administered simultaneously with the anti-PD-1 oranti-CTLA-4 antibody.

In certain embodiments, when administered on the same day, the anti-OX40antibody is administered about 15 to 45 minutes (e.g., about 30 minutes)before the anti-PD-1 or anti-CTLA-4 antibody. In certain embodiments,when administered on the same day, the anti-OX40 antibody isadministered about 15 to 45 minutes (e.g., about 30 minutes) after theanti-PD-1 or anti-CTLA-4 antibody.

Alternatively, anti-OX40 antibodies provided herein can be administeredat a flat dose (flat dose regimen).

In some cases, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, such that the dosage ofeach antibody administered falls within the ranges above. In addition,the antibodies usually are administered on multiple occasions.

Intervals between single dosages can be, for example, weekly, monthly,every three months or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml.

Anti-OX40 antibodies described herein may be administered with anotherantibody at the dosage regimen of the other antibody. For example, theanti-OX40 antibody may be administered with an anti-PD-1 antibody, suchas nivolumab (OPDIVO), every two weeks as an i.v. infusion over 60minutes until disease progression or unacceptable toxicity occurs.

Alternatively, the anti-OX40 antibody may be administered withpembrolizumab (KEYTRUDA) every 3 weeks as an i.v. infusion over 30minutes until disease progression or unacceptable toxicity occurs.

Antibodies can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

“Therapeutically effective dosages” of the antibodies described hereinpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. Inthe context of cancer, a therapeutically effective dose preferablyresults in increased survival, and/or prevention of furtherdeterioration of physical symptoms associated with cancer. Symptoms ofcancer are well-known in the art and include, for example, unusual molefeatures, a change in the appearance of a mole, including asymmetry,border, color and/or diameter, a newly pigmented skin area, an abnormalmole, darkened area under nail, breast lumps, nipple changes, breastcysts, breast pain, death, weight loss, weakness, excessive fatigue,difficulty eating, loss of appetite, chronic cough, worseningbreathlessness, coughing up blood, blood in the urine, blood in stool,nausea, vomiting, liver metastases, lung metastases, bone metastases,abdominal fullness, bloating, fluid in peritoneal cavity, vaginalbleeding, constipation, abdominal distension, perforation of colon,acute peritonitis (infection, fever, pain), pain, vomiting blood, heavysweating, fever, high blood pressure, anemia, diarrhea, jaundice,dizziness, chills, muscle spasms, colon metastases, lung metastases,bladder metastases, liver metastases, bone metastases, kidneymetastases, and pancreatic metastases, difficulty swallowing, and thelike.

A therapeutically effective dose may prevent or delay onset of cancer,such as may be desired when early or preliminary signs of the diseaseare present. Laboratory tests utilized in the diagnosis of cancerinclude chemistries (including the measurement of OX40 levels),hematology, serology and radiology. Accordingly, any clinical orbiochemical assay that monitors any of the foregoing may be used todetermine whether a particular treatment is a therapeutically effectivedose for treating cancer. One of ordinary skill in the art would be ableto determine such amounts based on such factors as the subject's size,the severity of the subject's symptoms, and the particular compositionor route of administration selected.

Antibodies and compositions described herein can be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Preferred routes of administration for antibodiesdescribed herein include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, the antibody can be administered via a non-parenteralroute, such as a topical, epidermal or mucosal route of administration,for example, intranasally, orally, vaginally, rectally, sublingually ortopically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Antibody compositions can be administered with medical devices known inthe art. For example, in one embodiment, the composition is administeredwith a needleless hypodermic injection device, such as the devicesdisclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants andmodules for use in administering the antibodies include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-OX40 antibodies are formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure theantibodies cross the BBB (if desired, e.g., for brain cancers), they canbe formulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein Areceptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p 120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273.

XVI. Uses and Methods

Anti-OX40 antibodies and compositions described herein have numerous invitro and in vivo applications involving, for example, enhancement ofimmune response by activating OX40 signaling, or detection of OX40. In apreferred embodiment, the antibodies are human antibodies. For example,anti-OX40 antibodies described herein can be contacted with cells inculture, in vitro or ex vivo, or administered to human subjects, e.g.,in vivo, to enhance immunity in a variety of diseases. Accordingly,provided herein are methods of modifying an immune response in a subjectcomprising administering to the subject an antibody, or antigen-bindingportion thereof, described herein such that the immune response in thesubject is modified. Preferably, the response is enhanced, stimulated orup-regulated.

Preferred subjects include human patients in whom enhancement of animmune response would be desirable. The methods are particularlysuitable for treating human patients having a disorder that can betreated by augmenting an immune response (e.g., a T-cell mediated immuneresponse, e.g., an antigen specific T cell response). In a particularembodiment, the methods are particularly suitable for treatment ofcancer in vivo. To achieve antigen-specific enhancement of immunity,anti-OX40 antibodies described herein can be administered together withan antigen of interest or the antigen may already be present in thesubject to be treated (e.g., a tumor-bearing or virus-bearing subject).When anti-OX40 antibodies are administered together with another agent,the two can be administered separately or simultaneously.

Also encompassed are methods for detecting the presence of human OX40antigen in a sample, or measuring the amount of human OX40 antigen,comprising contacting the sample, and a control sample, with anti-OX40antibodies (or antigen binding portions thereof) described herein, underconditions that allow for formation of a complex between the antibodyand human OX40. The formation of a complex is then detected, wherein adifference complex formation between the sample compared to the controlsample is indicative the presence of human OX40 antigen in the sample.The anti-OX40 antibodies described herein also can be used to purifyhuman OX40 via immunoaffinity purification.

Given the ability of anti-OX40 antibodies described herein to stimulateor co-stimulate T cell responses, e.g., antigen-specific T cellresponses, also provided herein are in vitro and in vivo methods ofusing the antibodies to stimulate, enhance or upregulateantigen-specific T cell responses, e.g., anti-tumor T cell responses. Incertain embodiments, CD3 stimulation is also included (e.g., bycoincubation with a cell expressing membrane CD3), which stimulation canbe provided at the same time, before, or after stimulation with ananti-OX40 antibody. In one embodiment, the method comprises contacting Tcells with an anti-OX40 antibody described herein, and optionally withan anti-CD3 antibody, such that an antigen-specific T cell response isstimulated. Any suitable indicator of an antigen-specific T cellresponse can be used to measure the antigen-specific T cell response.Non-limiting examples of such suitable indicators include increased Tcell proliferation in the presence of the antibody and/or increasecytokine production in the presence of the antibody. In a preferredembodiment, interleukin-2 and/or interferon-γ production by theantigen-specific T cell is stimulated.

T cells that can be enhanced or co-stimulated with anti-OX40 antibodiesinclude CD4+ T cells and CD8+ T cells. The T cells can be T_(eff) cells,e.g., CD4+ T_(eff) cells, CD8+ T_(eff) cells, Thelper (T_(h)) cells andT cytotoxic (T_(c)) cells.

Also provided are methods of stimulating an immune response (e.g., anantigen-specific T cell response) in a subject comprising administeringa therapeutically effective amount of an anti-OX40 antibody describedherein to the subject such that an immune response (e.g., anantigen-specific T cell response) in the subject is stimulated. In apreferred embodiment, the subject is a tumor-bearing subject and animmune response against the tumor is stimulated. A tumor may be a solidtumor or a liquid tumor, e.g., a hematological malignancy. In certainembodiments, a tumor is an immunogenic tumor. In certain embodiments, atumor is non-immunogenic. In certain embodiments, a tumor is PD-L1positive. In certain embodiments a tumor is PD-L1 negative. A subjectmay also be a virus-bearing subject and an immune response against thevirus is stimulated.

Further provided are methods for inhibiting growth of tumor cells in asubject comprising administering to the subject a therapeuticallyeffective amount of an anti-OX40 antibody described herein such thatgrowth of the tumor is inhibited in the subject. Also provided aremethods of treating viral infection in a subject comprisingadministering to the subject an anti-OX40 antibody described herein suchthat the viral infection is treated in the subject.

Also encompassed herein are methods for depleting Treg cells from thetumor microenvironment of a subject having a tumor, e.g., canceroustumor, comprising administering to the subject a therapeuticallyeffective amount of an anti-OX40 antibody described herein thatcomprises an Fc that stimulates depletion of T_(reg) cells in the tumormicroenvironment. An Fc may, e.g., be an Fc with effector function orenhanced effector function, such as binding or having enhanced bindingto one or more activating Fc receptors. In a preferred embodiment,T_(reg) depletion occurs without significant depletion or inhibition ofT_(eff) in the tumor microenvironment, and without significant depletionor inhibition of T_(eff) cells and T_(reg) cells outside of the tumormicroenvironment, e.g., in the periphery. In certain embodiments, thesubject has higher levels of OX40 on T_(reg) cells than on T_(eff)cells, e.g., in the tumor microenvironment.

In certain embodiments, the subject is treated with an anti-OX40antibody having an Fc that enhances agonism, e.g., binds to or hasenhanced binding to the inhibitory FcRIIb. Anti-OX40 antibodies maydeplete Tregs in tumors and/or Tregs in tumor infiltrating lymphocytes(TILs).

In certain embodiments, the anti-OX40 antibody is given to a subject asan adjunctive therapy. Treatments of subjects having cancer with theanti-OX40 antibody may lead to prolonged survival, e.g., long-termdurable response relative to the current standard of care; long termsurvival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 ormore years, or recurrence-free survival of at least 3 months, 6 months,9 months, 1, 2, 3, 4, 5, or 10 or more years. In certain embodiments,treatment of a subject having cancer with the anti-OX40 antibodyprevents recurrence of cancer or delays recurrence of cancer by, e.g., 3months, 6 months, 9 months, 1, 2, 3, 4, 5, or 10 or more years. Theanti-OX40 antibody treatment can be used as a first-, second-, orthird-line treatment.

In preferred embodiments, the anti-OX40 antibody is not significantlytoxic. For example, the antibody is not significantly toxic to an organof a human, e.g., one or more of the liver, kidney, brain, lungs, andheart, as determined, e.g., in clinical trials. In certain embodiments,the antibody does not significantly trigger an undesirable immuneresponse, e.g., autoimmunity or inflammation.

In certain embodiments, treatment of a subject with the anti-OX40antibody does not result in overstimulation of the immune system to theextent that the subject's immune system then attacks the subject itself(e.g., autoimmune response) or results in, e.g., anaphylaxis. Thus, theantibodies preferably do not cause anaphylaxis.

In certain embodiments, treatment of a subject with the anti-OX40antibody does not cause significant inflammatory reactions, e.g.,immune-mediated pneumonitis, immune-mediated colitis, immune mediatedhepatitis, immune-mediated nephritis or renal dysfunction,immune-mediated hypophysitis, immune-mediated hypothyroidism andhyperthyroidism, or other immune-mediated adverse reactions.

In certain embodiments, the anti-OX40 antibody provides synergisticanti-tumor effects in combination with another cancer therapy, such as acompound that stimulates the immune system (e.g., an immune-oncologyagent), e.g., a compound described herein or a compound modulating atarget described herein.

These and other methods described herein are discussed in further detailbelow.

Cancer

Activation of OX40 by anti-OX40 antibodies can enhance the immuneresponse to cancerous cells in the patient. Accordingly, provided hereinare methods for treating a subject having cancer, comprisingadministering to the subject the anti-OX40 antibodies described herein,such that the subject is treated, e.g., such that growth of canceroustumors is inhibited or reduced and/or that the tumors regress and/orthat prolonged survival is achieved. The anti-OX40 antibody can be usedalone to inhibit the growth of cancerous tumors. Alternatively, theanti-OX40 antibody can be used in conjunction with another agent, e.g.,another immunogenic agent, a standard cancer treatment, or anotherantibody, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of anti-OX40antibodies described herein. The antibody may be a human antibody.Additionally or alternatively, the antibody can be a chimeric orhumanized antibody.

Also provided herein are combination therapies comprising administrationof an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody totreat subjects having tumors (e.g., advanced solid tumors).

In certain embodiments, provided herein are methods of treating cancerwherein an anti-OX40 antibody and an anti-PD-1 antibody or anti-CTLA-4antibody are administered to a patient with a tumor (e.g., advancedsolid tumor) according to a defined clinical dosage regimen. In certainembodiments, the anti-OX40 antibody is OX40.21. In certain embodiments,the anti-PD-1 antibody is BMS-936558 (nivolumab). In certainembodiments, the anti-CTLA-4 antibody is ipilimumab (Yervoy®). Incertain embodiments, dosage regimens are adjusted to provide the optimumdesired response (e.g., an effective response).

As used herein, adjunctive or combined administration (coadministration)includes simultaneous administration of the compounds in the same ordifferent dosage form, or separate administration of the compounds(e.g., sequential administration). Thus, the anti-OX40 and anti-PD-1antibody or anti-CTLA-4 antibody can be simultaneously administered in asingle formulation. Alternatively, the anti-OX40 and anti-PD-1 antibodyor anti-CTLA-4 antibody can be formulated for separate administrationand are administered concurrently or sequentially (e.g., one antibody isadministered within about 30 minutes prior to administration of thesecond antibody).

For example, the anti-PD1 antibody or anti-CTLA-4 antibody can beadministered first and followed by (e.g., immediately followed by) theadministration of the anti-OX40 antibody, or vice versa. In certainembodiments, the anti-PD-1 antibody or anti-CTLA-4 antibody isadministered prior to administration of the anti-OX40 antibody. Inanother embodiment, the anti-PD-1 antibody or anti-CTLA-4 antibody isadministered after administration of the anti-OX40 antibody. In anotherembodiment, the anti-OX40 antibody and anti-PD-1 antibody or anti-CTLA-4antibody are administered concurrently. Such concurrent or sequentialadministration preferably results in both antibodies beingsimultaneously present in treated patients.

Cancers whose growth may be inhibited with anti-OX40 antibodies, orcombination therapy with an anti-OX40 and an anti-PD-1 or anti-CTLA-4antibody, include cancers typically responsive to immunotherapy andthose that are not typically responsive to immunotherapy. Cancers may becancers with solid tumors or blood malignancies (liquid tumors).Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non squamous NSCLC, glioma,gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma),ovarian cancer, liver cancer, colorectal cancer, endometrial cancer,kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g.hormone refractory prostate adenocarcinoma), thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma (glioblastomamultiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer (or carcinoma),gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal naturalkiller, melanoma (e.g., metastatic malignant melanoma, such as cutaneousor intraocular malignant melanoma), bone cancer, skin cancer, uterinecancer, cancer of the anal region, testicular cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumors of childhood, cancer of the ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,squamous cell cancer, T-cell lymphoma, environmentally-induced cancersincluding those induced by asbestos, virus-related cancers or cancers ofviral origin (e.g., human papilloma virus (HPV-related or -originatingtumors)), and hematologic malignancies derived from either of the twomajor blood cell lineages, i.e., the myeloid cell line (which producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells) orlymphoid cell line (which produces B, T, NK and plasma cells), such asall types of leukemias, lymphomas, and myelomas, e.g., acute, chronic,lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),and chronic myelogenous leukemia (CML), undifferentiated AML (M0),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cellhematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas,lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma,mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle celllymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma,intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein may also be used for treatment of metastaticcancers, unresectable and/or refractory cancers (e.g., cancersrefractory to previous immunotherapy, e.g., with a blocking CTLA-4 orPD-1 antibody), and recurrent cancers.

In certain embodiments, the patient being treated with the anti-OX40antibody, or combination of anti-OX40 antibody and anti-PD-1 oranti-CTLA-4 antibody, has an advanced solid tumor. For example, in oneembodiment, the patient to be treated has cervical cancer. In anotherembodiment, the patient to be treated has colorectal (CRC) cancer. Inanother embodiment, the patient to be treated has bladder cancer (e.g.,unresectable locally advanced or metastatic bladder cancer). In anotherembodiment, the patient to be treated has ovarian cancer (e.g.,unresectable locally advanced or metastatic ovarian cancer).

In one embodiment, the patient being treated with the anti-OX40antibody, or combination of anti-OX40 antibody and anti-PD-1 oranti-CTLA-4 antibody, has non-small cell lung cancer (NSCLC). In anotherembodiment, the patient to be treated has squamous cell carcinoma of thehead and neck (SCCHN). In another embodiment, the patient to be treatedhas B-cell non-Hodgkin's lymphoma (B-NHL). In another embodiment, thepatient to be treated has myeloma. In another embodiment, the patienthas melanoma. In another embodiment, the patient to be treated hasdiffuse large B-cell lymphoma (DLBCL).

In certain embodiments, the anti-OX40 antibody is administered topatients having a cancer that exhibited an inadequate response to aprior treatment, e.g., a prior treatment with an immuno-oncology drug,or patients having a cancer that is refractory or resistant, eitherintrinsically refractory or resistant (e.g., refractory to a PD-1pathway antagonist), or a wherein the resistance or refractory state isacquired. For example, subjects who are not responsive or notsufficiently responsive to a first therapy or who see diseaseprogression following treatment, e.g., anti-PD-1 treatment, may betreated by administration of the anti-OX40 antibody alone or incombination with another therapy (e.g., with an anti-PD-1 therapy).

In certain embodiments, the anti-OX40 antibody is administered topatients who have not previously received (i.e., been treated with) animmuno-oncology agent, e.g., a PD-1 pathway antagonist.

In certain embodiments, the anti-OX40 antibody may be administered witha standard of care treatment (e.g., surgery, radiation, andchemotherapy). In other embodiments, the anti-OX40 antibody may beadministered as a maintenance therapy, e.g., a therapy that is intendedto prevent the occurrence or recurrence of tumors.

In certain embodiments, the anti-OX40 antibody may be administered withanother treatment, e.g., radiation, surgery, or chemotherapy. Forexample, anti-OX40 antibody adjunctive therapy may be administered whenthere is a risk that micrometastases may be present and/or in order toreduce the risk of a relapse.

In certain embodiments, the anti-OX40 antibody can be administered as amonotherapy, or as the only immunostimulating therapy. In otherembodiments, the anti-OX40 antibody can also be combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al (2004) J. Immunol. 173:4919-28). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By lowering the threshold of T cell activation via OX40activation, the tumor responses in the host can be activated, allowingtreatment of non-immunogenic tumors or those having limitedimmunogenicity.

In some embodiments, the anti-OX40 antibody can be used in conjunctionwith a vaccination protocol. Many experimental strategies forvaccination against tumors have been devised (see Rosenberg, S., 2000,Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62;Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D.2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCOEducational Book Spring: 730-738; see also Restifo, N. and Sznol, M.,Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997,Cancer: Principles and Practice of Oncology, Fifth Edition). In one suchstrategy, of these strategies, a vaccine is prepared using autologous orallogeneic tumor cells. These cellular vaccines have been shown to bemost effective when the tumor cells are transduced to express GM-CSF.GM-CSF has been shown to be a potent activator of antigen presentationfor tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. SciU.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. OX40 activation can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with OX40activation is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withOX40 activation to activate more potent anti-tumor responses.

Anti-OX40 antibodies described herein can also be combined withchemotherapeutic regimes. In these instances, it may be possible toreduce the dose of chemotherapeutic reagent administered (Mokyr et al.(1998) Cancer Research 58: 5301-5304). For example, the anti-OX40antibody can be used in combination with decarbazine to treat melanoma.In another example, the anti-OX40 antibody can be used in combinationwith interleukin-2 (IL-2) to treat melanoma. The scientific rationalebehind the combined use of anti-OX40 antibodies and chemotherapy is thatcell death, a consequence of the cytotoxic action of mostchemotherapeutic compounds, should result in increased levels of tumorantigen in the antigen presentation pathway. Other combination therapiesthat may result in synergy with anti-OX40 antibodies through cell deathare radiation, surgery, and hormone deprivation. Each of these protocolscreates a source of tumor antigen in the host. Angiogenesis inhibitorscan also be used in combination with the anti-OX40 antibody. Inhibitionof angiogenesis leads to tumor cell death which may feed tumor antigeninto host antigen presentation pathways.

Anti-OX40 antibodies described herein can also be used in combinationwith bispecific antibodies that target Fcα or Fcγ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would be augmentedby the activation of OX40. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-OX40 antibodies to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with the anti-OX40 antibodies described herein. Theseinclude molecules on the surface of dendritic cells which activate DCfunction and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity (Ridge et al. (1998)Nature 393: 474-478) and can be used in conjunction with anti-OX40antibodies. Activating antibodies to T cell costimulatory molecules suchas CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000)Immunol 164: 2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3:682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) mayalso provide for increased levels of T cell activation. Inhibitors ofPD1 or PD-L1 may also be used in conjunction with anti-OX40 antibodies.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. Anti-OX40 antibodies can be used to increasethe effectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Er vivo activation in thepresence of anti-OX40 antibodies can increase the frequency and activityof the adoptively transferred T cells.

Infectious Diseases

Also provided herein are methods to treat patients who have been exposedto particular toxins or pathogens. Accordingly, provided herein aremethods of treating an infectious disease in a subject comprisingadministering to the subject anti-OX40 antibodies described herein, suchthat the subject is treated for the infectious disease. In certainembodiments, the anti-OX40 antibody is a chimeric or humanized antibody.

Similar to its application to tumors as discussed above, anti-OX40antibodies can be used alone, or as an adjuvant, in combination withvaccines, to stimulate the immune response to pathogens, toxins, andself-antigens. Examples of pathogens for which this therapeutic approachcan be particularly useful, include pathogens for which there iscurrently no effective vaccine, or pathogens for which conventionalvaccines are less than completely effective. These include, but are notlimited to HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia,Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.Anti-OX40 antibodies may be useful against established infections byagents such as HIV that present altered antigens over the course of theinfections. These novel epitopes are recognized as foreign at the timeof anti-OX40 antibody administration, thus provoking a strong T cellresponse.

Some examples of pathogenic viruses causing infections treatable by themethods described herein include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable by themethods described herein include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable by themethods described herein include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bythe methods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, anti-OX40 antibodies can be combined withother forms of immunotherapy such as cytokine treatment (e.g.,interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, whichprovides for enhanced presentation of tumor antigens (see, e.g.,Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994)Structure 2:1121-1123).

Autoimmune Reactions

Anti-OX40 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489;Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987;Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4). Therefore, anti-OX40 antibodies can be usedin conjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of Aβ peptide in amyloid deposits inthe brain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFc for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-OX40 antibodies. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-OX40 antibodiescan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

Vaccines

The anti-OX40 antibodies described herein can be used to stimulateantigen-specific immune responses by coadministration of the antibodieswith an antigen of interest (e.g., a vaccine). Accordingly, providedherein are methods of enhancing an immune response to an antigen in asubject, comprising administering to the subject: (i) the antigen; and(ii) an anti-OX40 antibody such that an immune response to the antigenin the subject is enhanced. The antibody may be a human anti-OX40antibody (such as any of the human anti-OX40 antibodies describedherein). In other embodiments, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

In certain embodiments, a peptide or fusion protein comprising theepitope to which the anti-OX40 antibody binds is used as a vaccineinstead of, or in addition to, the anti-OX40 antibody.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, anti-OX40 antibodies described herein can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of anti-OX40antibodies, or antigen binding fragments thereof, described herein withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can address problems related to thedevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells which would render them unreactive with the antibody.

Also provided herein are kits comprising the anti-OX40 antibodycompositions described herein (e.g., human antibodies, bispecific ormultispecific molecules, or immunoconjugates) and instructions for use.The kit can further contain at least one additional reagent, or one ormore additional human antibodies described herein (e.g., a humanantibody having a complementary activity which binds to an epitope inOX40 distinct from the first human antibody). Kits typically include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit.

Treatment Protocols

Suitable protocols for treating a solid tumor (e.g., an advanced solidtumor) in a human patient include, for example, administering to thepatient an effective amount of an anti-OX40 antibody comprising CDR1,CDR2 and CDR3 domains of the heavy chain variable region having thesequence set forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains ofthe light chain variable region having the sequence set forth in SEQ IDNO: 94, wherein the method comprises at least one administration cycle,wherein the cycle is a period of two weeks (Q2W), wherein for each ofthe at least one cycles, at least one dose of the anti-OX40 antibody isadministered at a dose of 1 mg/kg body weight; a fixed dose of 20, 40,80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or a fixed doseof about 20, 40, 80, 160, or 320 mg.

Another suitable protocol for treating a solid tumor in a human patientincludes, for example, administering to the patient an effective amountof each of:

(a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:318, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94, and

(b) an anti-PD-1 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:301, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 302,

wherein the method comprises at least one administration cycle, whereinthe cycle is a period of two weeks, wherein for each of the at least onecycles, at least one dose of the anti-OX40 antibody is administered at adose of 1 mg/kg body weight; a fixed dose of 20, 40, 80, 160, or 320 mg;a dose of about 1 mg/kg body weight; or a fixed dose of about 20, 40,80, 160, or 320 mg, and at least one dose of the anti-PD-1 antibody isadministered at flat dose of 240 mg or a flat dose of about 240 mg. Insome embodiments, the anti-PD-1 antibody is administered once everythree weeks (q3w) at a fixed dose of 360 mg, or once every four weeks(q4w) at a dose of 480 mg.

Another suitable protocol for treating a solid tumor in a human patientincludes, for example, administering to the patient an effective amountof each of:

(a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:318, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94, and

(b) an anti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3 domains ofthe heavy chain variable region having the sequence set forth in SEQ IDNO: 309, and CDR1, CDR2 and CDR3 domains of the light chain variableregion having the sequence set forth in SEQ ID NO: 310,

wherein the method comprises at least one administration cycle, whereinthe cycle is a period of three weeks (q3w), wherein for each of the atleast one cycles, at least one dose of the anti-OX40 antibody isadministered at a dose of 1 mg/kg body weight; a fixed dose of 20, 40,80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or a fixed doseof about 20, 40, 80, 160, or 320 mg, and at least one dose of theanti-CTLA-4 antibody is administered at flat dose of 1 mg/kg body weightor a flat dose of about 1 mg/kg body weight. In one embodiment, theanti-OX40 antibody is administered together with the anti-CTLA-4antibody for at least one cycle, followed by anti-OX40 antibodymonotherapy for at least one cycle. In certain embodiments, theanti-OX40 antibody is administered together with ipilimumab for theinitial four cycles, followed by anti-OX40 antibody monotherapy forsubsequent cycles.

In some embodiments, the anti-OX40 antibody and anti-PD-1 antibody areadministered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg ofanti-PD-1 antibody;

(b) 20 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(c) 40 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(d) 80 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(e) 160 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody; or

(f) 320 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody.

In some embodiments, the anti-OX40 antibody and anti-CTLA-4 antibody areadministered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody; or

(f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody.

In one embodiment, the dose of the anti-OX40 and/or anti-PD-1 oranti-CTLA-4 antibody is calculated per body weight, e.g., mg/kg bodyweight. In another embodiment, the dose of the anti-OX40 and/oranti-PD-1 or anti-CTLA-4 antibody is a flat-fixed dose. In anotherembodiment, the dose of the anti-OX40 and/or anti-PD-1 or anti-CTLA-4antibody is varied over time. For example, the anti-OX40 and/oranti-PD-1 or anti-CTLA-4 antibody may be initially administered at ahigh dose and may be lowered over time. In another embodiment, theanti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is initiallyadministered at a low dose and increased over time.

In another embodiment, the amount of the anti-OX40 and/or anti-PD-1 oranti-CTLA-4 antibody administered is constant for each dose. In anotherembodiment, the amount of antibody administered varies with each dose.For example, the maintenance (or follow-on) dose of the antibody can behigher or the same as the loading dose which is first administered. Inanother embodiment, the maintenance dose of the antibody can be lower orthe same as the loading dose.

In some embodiments, the anti-OX40 and/or anti-PD-1 or anti-CTLA-4antibody are formulated for intravenous administration. In someembodiments, the anti-OX40 antibody, or anti-OX40 antibody and anti-PD-1or CTLA-4 antibody, are administered on Day 1 of each cycle.

In some embodiments, the anti-OX40 and/or anti-PD-1 or anti-CTLA-4antibody are administered once per week, once every two weeks, onceevery three weeks, or once every four weeks, or as long as a clinicalbenefit is observed or until there is a complete response, confirmedprogressive disease or unmanageable toxicity.

In one embodiment, a cycle of administration is two weeks, which can berepeated, as necessary. In another embodiment, the cycle is three weeks.In some embodiments, the treatment consists of up to eight cycles. Inother embodiments, the treatment consists of up to 12 cycles.

In one embodiment, one dose each of an anti-OX40 antibody and ananti-PD-1 antibody is administered per two week cycle. In anotherembodiment, one dose each of the anti-PD-1 antibody and anti-OX40antibody is administered per three week cycle. In another embodiment,one dose each of the anti-PD-1 antibody and anti-OX40 antibody isadministered per four week cycle.

In one embodiment, one dose each of the anti-OX40 antibody andanti-CTLA-4 antibody is administered per three week cycle. In someembodiments, one dose each of the anti-OX40 antibody and anti-CTLA-4antibody is administered per three week cycle for the first four cycles,followed by anti-OX40 antibody monotherapy for the fifth through eighthcycles.

In another embodiment, the anti-OX40 antibody and anti-PD-1 oranti-CTLA-4 antibody are administered as a first line of treatment(e.g., the initial or first treatment). In another embodiment, theanti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody areadministered as a second line of treatment (e.g., after the initial orfirst treatment, including after relapse and/or where the firsttreatment has failed).

In another aspect, the invention features any of the aforementionedembodiments, wherein the anti-PD-1 antibody is replaced by, or combinedwith, an anti-PD-L1 or anti-PD-L2 antibody.

In some embodiments, the human patient has a cancer selected from thegroup consisting of cervical cancer, bladder cancer, colorectal cancer,and ovarian cancer.

In certain embodiments, the anti-OX40 antibody comprises a heavy chainvariable region CDR1 comprising the sequence set forth in SEQ ID NO: 87,a heavy chain variable region CDR2 comprising the sequence set forth inSEQ ID NO: 317, a heavy chain variable region CDR3 comprising thesequence set forth in SEQ ID NO: 89, a light chain variable region CDR1comprising the sequence set forth in SEQ ID NO: 90, a light chainvariable region CDR2 comprising the sequence set forth in SEQ ID NO: 91,and a light chain variable region CDR3 comprising the sequence set forthin SEQ ID NO: 92. In certain embodiments, the anti-OX40 antibodycomprises heavy and light chain variable regions comprising thesequences set forth in SEQ ID NOs: 318 and 94, respectively. In certainembodiments, the anti-OX40 antibody comprises heavy and light chainsequences comprising the sequences set forth in SEQ ID NOs: 124 and 116,respectively.

In certain embodiments, the anti-PD-1 antibody comprises a heavy chainvariable region CDR1, CDR2, and CDR3 comprising the sequences set forthin SEQ ID NOs: 303-305, respectively, and light chain variable regionCDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:306-308, respectively. In certain embodiments, the anti-PD-1 antibodycomprises heavy and light chain variable regions sequences set forth inSEQ ID NOs: 301 and 302, respectively. In certain embodiments, theanti-PD-1 antibody comprises heavy and light chain sequences set forthin SEQ ID NOs: 299 and 300, respectively.

In certain embodiments, the anti-CTLA-4 antibody comprises a heavy chainvariable region CDR1, CDR2, and CDR3 comprising the sequences set forthin SEQ ID NOs: 311-313, respectively, and light chain variable regionCDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:314-316, respectively. In certain embodiments, the anti-CTLA-4 antibodycomprises heavy and light chain variable regions sequences set forth inSEQ ID NOs: 309 and 310, respectively.

Outcomes

With respect to target lesions, responses to therapy may include:

Complete Response (CR) Disappearance of all target lesions. Any (RECISTV1.1) pathological lymph nodes (whether target or non-target) must havereduction in short axis to <10 mm. Partial Response (PR) At least a 30%decrease in the sum of the (RECIST V1.1) diameters of target lesions,taking as reference the baseline sum diameters. Progressive Disease (PD)At least a 20% increase in the sum of the (RECIST V1.1) diameters oftarget lesions, taking as reference the smallest sum on study (thisincludes the baseline sum if that is the smallest on study). In additionto the relative increase of 20%, the sum must also demonstrate anabsolute increase of at least 5 mm. (Note: the appearance of one or morenew lesions is also considered progression). Stable Disease (SD) Neithersufficient shrinkage to qualify for (RECIST V1.1) PR nor sufficientincrease to qualify for PD, taking as reference the smallest sumdiameters while on study. Immune-related Disappearance of all targetlesions. Any Complete Response (irCR) pathological lymph nodes (whethertarget (irRECIST) or non-target) must have reduction in short axis to<10 mm. Immune-related At least a 30% decrease in the sum of PartialResponse (irPR) diameters of target lesions and all new (irRECIST)measurable lesions (ie Percentage Change in Tumor Burden), taking asreference the baseline sum diameters. Note: the appearance of newmeasurable lesions is factored into the overall Tumor Burden, but doesnot automatically qualify as progressive disease until the sum of thediameters increases by ≥20% when compared to nadir. Immune-related Atleast a 20% increase in Tumor Burden Progressive Disease (irPD) (ie thesum of diameters of target lesions, (irRECIST) and any new measurablelesions) taking as reference the smallest sum on study (this includesthe baseline sum if that is the smallest on study). In addition to therelative increase of 20%, the sum must also demonstrate an absoluteincrease of at least 5 mm. Tumor assessments using immune- relatedcriteria for progressive disease incorporates the contribution of newmeasurable lesions. Each net percentage change in tumor burden perassessment accounts for the size and growth kinetics of both old and newlesions as they appear. Immune-related Neither sufficient shrinkage toqualify for Stable Disease (irSD) irPR nor sufficient increase toqualify for (irRECIST) irPD, taking as reference the smallest sumdiameters while on study.

With respect to non-target lesions, responses to therapy may include:

Complete Response (CR) Disappearance of all non-target lesions. (RECISTV1.1) All lymph nodes must be non-pathological in size (<10 mm shortaxis). Non-CR/Non-PD Persistence of one or more non-target (RECIST V1.1)lesion(s). Progressive Disease (PD) Unequivocal progression of existingnon- (RECIST V1.1) target lesions. The appearance of one or more newlesions is also considered progression. Immune-related Disappearance ofall non-target lesions. All Complete Response (irCR) lymph nodes must benon-pathological in (irRECIST) size (<10 mm short axis). Immune-relatedIncreases in number or size of non-target Progressive Disease (irPD)lesion(s) does not constitute progressive (irRECIST) diseaseunless/until Tumor Burden increases by 20% (ie the sum of the diametersat nadir of target lesions and any new measurable lesions increases bythe required amount). Non-target lesions are not considered in thedefinition of Stable Disease and Partial Response.

Patients treated according to the methods disclosed herein preferablyexperience improvement in at least one sign of cancer. In oneembodiment, improvement is measured by a reduction in the quantityand/or size of measurable tumor lesions. In another embodiment, lesionscan be measured on chest x-rays or CT or MRI films. In anotherembodiment, cytology or histology can be used to evaluate responsivenessto a therapy.

In one embodiment, the patient treated exhibits a complete response(CR), a partial response (PR), stable disease (SD), immune-relatedcomplete disease (irCR), immune-related partial response (irPR), orimmune-related stable disease (irSD). In another embodiment, the patienttreated experiences tumor shrinkage and/or decrease in growth rate,i.e., suppression of tumor growth. In another embodiment, unwanted cellproliferation is reduced or inhibited. In yet another embodiment, one ormore of the following can occur: the number of cancer cells can bereduced; tumor size can be reduced; cancer cell infiltration intoperipheral organs can be inhibited, retarded, slowed, or stopped; tumormetastasis can be slowed or inhibited; tumor growth can be inhibited;recurrence of tumor can be prevented or delayed; one or more of thesymptoms associated with cancer can be relieved to some extent.

In other embodiments, administration of effective amounts of theanti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody according toany of the methods provided herein produces at least one therapeuticeffect selected from the group consisting of reduction in size of atumor, reduction in number of metastatic lesions appearing over time,complete remission, partial remission, or stable disease. In still otherembodiments, the methods of treatment produce a comparable clinicalbenefit rate (CBR=CR+PR+SD ≥6 months) better than that achieved by ananti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody alone. In otherembodiments, the improvement of clinical benefit rate is about 20% 20%,30%, 40%, 50%, 60%, 70%, 80% or more compared to an anti-OX40 antibodyor anti-PD-1 or anti-CTLA-4 antibody alone.

Combination Therapies

In addition to the combinations therapies provided above, anti-OX40antibodies described herein can be used in combination therapy, asdescribed below.

Methods of combination therapy include those in which an anti-OX40antibody, or a combination of anti-OX40 antibody and anti-PD-1 oranti-CTLA-4 antibody, is coadministered with one or more additionalagents, e.g., small molecule drugs, antibodies or antigen bindingportions thereof, and which are effective in stimulating immuneresponses to thereby further enhance, stimulate or upregulate immuneresponses in a subject. For instance, as shown in the Examples, theadministration of an anti-OX40 antibody and an antagonist anti-PD-1antibody to mice can result in a synergic effect in inhibiting tumorgrowth.

The anti-OX40 antibody can be combined with (i) an agonist of astimulatory (e.g., co-stimulatory) molecule (e.g., receptor or ligand)and/or (ii) an antagonist of an inhibitory signal or molecule (e.g.,receptor or ligand) on immune cells, such as T cells, both of whichresult in amplifying immune responses, such as antigen-specific T cellresponses. In certain aspects, an immuno-oncology agent is (i) anagonist of a stimulatory (including a co-stimulatory) molecule (e.g.,receptor or ligand) or (ii) an antagonist of an inhibitory (including aco-inhibitory) molecule (e.g., receptor or ligand) on cells involved ininnate immunity, e.g., NK cells, and wherein the immuno-oncology agentenhances innate immunity. Such immuno-oncology agents are often referredto as immune checkpoint regulators, e.g., immune checkpoint inhibitor orimmune checkpoint stimulator.

In certain embodiments, the anti-OX40 antibody is administered with anagent that targets a stimulatory or inhibitory molecule that is a memberof the immunoglobulin super family (IgSF). For example, the anti-OX40antibody may be administered to a subject with an agent that targets amember of the IgSF family to increase an immune response. In otherembodiments, the anti-OX40 antibody may be administered with an agentthat targets (or binds specifically to) a member of the B7 family ofmembrane-bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC(PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or aco-stimulatory or co-inhibitory receptor binding specifically to a B7family member.

The anti-OX40 antibody may also be administered with an agent thattargets a member of the TNF and TNFR family of molecules (ligands orreceptors), such as CD40 and CD40L, GITR, GITR-L, CD70, CD27L, CD30,CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3,TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI,APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2,TNFR1, Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS,FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) Drug DiscoveryToday 00:1).

T cell responses can be stimulated by a combination of anti-OX40antibodies and one or more of the following agents:

-   -   (1) An antagonist (inhibitor or blocking agent) of a protein        that inhibits T cell activation (e.g., immune checkpoint        inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as        described above, and any of the following proteins: TIM-3,        Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113,        GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1,        and TIM-4; and/or    -   (2) An agonist of a protein that stimulates T cell activation,        such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L,        GITR, GITR-L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with the anti-OX40 antibody for treating cancer, include:Yervoy™ (ipilimumab) or Tremelimumab (to CTLA-4), galiximab (to B7.1),BMS-936558 (to PD-1), MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559(to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2),MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566(to CD137), CDX-1127 (to CD27), Atacicept (to TACI), CP-870893 (toCD40), Lucatumumab (to CD40), Dacetuzumab (to CD40), Muromonab-CD3 (toCD3), Ipilumumab (to CTLA-4).

Anti-OX40 antibodies may also be administered with pidilizumab (CT-011).

Other molecules that can be combined with the anti-OX40 antibody for thetreatment of cancer include antagonists of inhibitory receptors on NKcells or agonists of activating receptors on NK cells. For example, theanti-OX40 antibody can be combined with antagonists of KIR (e.g.,lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-OX40antibodies may be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In certain embodiments, anti-OX40 antibodies can be used in combinationwith (i) antagonists (or inhibitors or blocking agents) of proteins ofthe IgSF family or B7 family or the TNF family that inhibit T cellactivation or antagonists of cytokines that inhibit T cell activation(e.g., IL-6, IL-10, TGF-B, VEGF; “immunosuppressive cytokines”) and/or(ii) agonists of stimulatory receptors of the IgSF family, B7 family orthe TNF family or of cytokines that stimulate T cell activation, forstimulating an immune response, e.g., for treating proliferativediseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

The anti-OX40 antibodies may also be administered with agents thatinhibit TGF-β signaling.

Additional agents that may be combined with the anti-OX40 antibodiesdescribed herein include agents that enhance tumor antigen presentation,e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpGoligonucleotides, and imiquimod, or therapies that enhance theimmunogenicity of tumor cells (e.g., anthracyclines).

Yet other therapies that may be combined with the anti-OX40 antibodiesinclude therapies that deplete or block Treg cells, e.g., an agent thatspecifically binds to CD25.

Another therapy that may be combined with the anti-OX40 antibodies is atherapy that inhibits a metabolic enzyme such as indoleamine dioxigenase(IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that may be used with the anti-OX40 antibodiesincludes agents that inhibit the formation of adenosine or inhibit theadenosine A2A receptor.

Other therapies that may be combined with anti-OX40 antibodies fortreating cancer include therapies that reverse/prevent T cell anergy orexhaustion and therapies that trigger an innate immune activation and/orinflammation at a tumor site.

The anti-OX40 antibody may be combined with more than oneimmuno-oncology agent, and may be, e.g., combined with a combinatorialapproach that targets multiple elements of the immune pathway, such asone or more of the following: a therapy that enhances tumor antigenpresentation (e.g., dendritic cell vaccine, GM-CSF secreting cellularvaccines, CpG oligonucleotides, imiquimod); a therapy that inhibitsnegative immune regulation e.g., by inhibiting CTLA-4 and/orPD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or otherimmune suppressing cells; a therapy that stimulates positive immuneregulation, e.g., with agonists that stimulate the CD-137 and/or GITRpathway and/or stimulate T cell effector function; a therapy thatincreases systemically the frequency of anti-tumor T cells; a therapythat depletes or inhibits Tregs, such as Tregs in the tumor, e.g., usingan antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion; a therapy that impacts the function of suppressor myeloidcells in the tumor; a therapy that enhances immunogenicity of tumorcells (e.g., anthracyclines); adoptive T cell or NK cell transferincluding genetically modified cells, e.g., cells modified by chimericantigen receptors (CAR-T therapy); a therapy that inhibits a metabolicenzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, ornitric oxide synthetase; a therapy that reverses/prevents T cell anergyor exhaustion; a therapy that triggers an innate immune activationand/or inflammation at a tumor site; administration of immunestimulatory cytokines; or blocking of immuno repressive cytokines.

Anti-OX40 antibodies can be used together with one or more of agonisticagents that ligate positive costimulatory receptors, blocking agentsthat attenuate signaling through inhibitory receptors, antagonists, andone or more agents that increase systemically the frequency ofanti-tumor T cells, agents that overcome distinct immune suppressivepathways within the tumor microenvironment (e.g., block inhibitoryreceptor engagement (e.g., PD-L1/PD-1 interactions), deplete or inhibitTregs (e.g., using an anti-CD25 monoclonal antibody (e.g., daclizumab)or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes suchas IDO, or reverse/prevent T cell anergy or exhaustion) and agents thattrigger innate immune activation and/or inflammation at tumor sites.

In certain embodiments, the anti-OX40 antibody is administered to asubject together with a BRAF inhibitor if the subject is BRAF V600mutation positive.

In certain embodiments, the anti-OX40 antibody is administered togetherwith another immunostimulatory antibody.

Provided herein are methods for stimulating an immune response in asubject comprising administering to the subject the anti-OX40 antibody,and one or more additional immunostimulatory antibodies, such as ananti-PD-1 antagonist, e.g., antagonist antibody, an anti-PD-L1antagonist, e.g., antagonist antibody, an antagonist anti-CTLA-4antagonist, e.g., antagonist antibody and/or an anti-LAG3 antagonist,e.g., an antagonist antibody, such that an immune response is stimulatedin the subject, for example to inhibit tumor growth or to stimulate ananti-viral response. In one embodiment, the subject is administered theanti-OX40 antibody and an antagonist anti-PD-1 antibody. In oneembodiment, the subject is administered the anti-OX40 antibody and anantagonist anti-PD-L1 antibody. In one embodiment, the subject isadministered the anti-OX40 antibody and an antagonist anti-CTLA-4antibody. In one embodiment, the anti-OX40 antibody is a human antibody.Alternatively, the anti-OX40 antibody can be, for example, a chimeric orhumanized antibody. In one embodiment, the at least one additionalimmunostimulatory antibody (e.g., an antagonist anti-PD-1, an antagonistanti-PD-L1, an antagonist anti-CTLA-4 and/or an antagonist anti-LAG3antibody) is a human antibody. Alternatively, the at least oneadditional immunostimulatory antibody can be, for example, a chimeric orhumanized antibody (e.g., prepared from a mouse anti-PD-1, anti-PD-L1,anti-CTLA-4 and/or anti-LAG3 antibody).

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering the anti-OX40 antibody with anantagonist PD-1 antibody, an antagonist PD-L1 antibody, an anti-CTLA-4antibody, or an anti-LAG3 antibody to a subject. In certain embodiments,one or both antibodies are administered at a subtherapeutic dose. Alsoprovided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease with an immunostimulatoryagent, comprising administering the anti-OX40 antibody and asubtherapeutic dose of an anti-PD-1, anti-PD-L1, anti-CTLA-4, oranti-LAG3 antibody to a subject (e.g., a human). In certain embodiments,the anti-OX40 antibody comprises the CDRs or variable regions of 3F4,14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2,and 20C1, or is another agonist anti-OX40 antibody described herein.

Suitable PD-1 antagonists for use in the methods described herein,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusionprotein, such as AMP-244. In one embodiment, the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibodythat comprises the CDRs or variable regions of one of antibodies 17D8,2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certainembodiments, an anti-PD1 antibody is MK-3475 (Lambrolizumab) describedin WO2012/145493; and AMP-514 described in WO 2012/145493. Further knownPD-1 antibodies and other PD-1 inhibitors include those described in WO2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149, andU.S. Patent Publication No. 2009/0317368. Any of the anti-PD-1antibodies disclosed in WO2013/173223 may also be used. An anti-PD-1antibody that competes for binding with, and/or binds to the sameepitope on PD-1 as, as one of these antibodies may also be used incombination treatments. Another approach to target the PD-1 receptor isthe recombinant protein composed of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgG1, called AMP-224. In certainembodiments, the antibody has at least about 90% variable region aminoacid sequence identity with the above-mentioned antibodies.

In certain embodiments, the anti-OX40 antibody is used in combinationwith nivolumab, which comprises heavy and light chains comprising thesequences shown in SEQ ID NOs: 299 and 300, respectively, or antigenbinding fragments and variants thereof. In certain embodiments, theantibody has heavy and light chain CDRs or variable regions ofnivolumab. Accordingly, in one embodiment, the antibody comprises CDR1,CDR2, and CDR3 domains of the VH of nivolumab having the sequence setforth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3 domains of the VL ofnivolumab having the sequence set forth in SEQ ID NO: 302. In certainembodiments, the antibody comprises CDR1, CDR2 and CDR3 domainscomprising the sequences set forth in SEQ ID NOs: 303-305, respectively,and CDR1, CDR2 and CDR3 domains comprising the sequences set forth inSEQ ID NOs: 306-308, respectively. In certain embodiments, the antibodycomprises VH and/or VL regions comprising the amino acid sequences setforth in SEQ ID NO: 301 and/or SEQ ID NO: 302, respectively. In certainembodiments, the antibody has at least about 90%, e.g., at least about90%, 95%, or 99% variable region identity with SEQ ID NO: 301 or SEQ IDNO: 302.

Exemplary anti-PD-L1 antibodies include BMS-936559 (referred to as 12A4in WO 2007/005874 and U.S. Pat. No. 7,943,743), or an antibody thatcomprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005874 and U.S. Pat. No. 7,943,743. In certain embodiments, theanti-PD-L1 antibody is MEDI4736 (also known as Anti-B7-H1), MPDL3280A(also known as RG7446), MSB0010718C (WO2013/79174), or rHigM12B7. Any ofthe anti-PD-L1 antibodies disclosed in WO2013/173223, WO2011/066389,WO2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S.Publication No. 2009/145493 may also be used.

Exemplary anti-CTLA-4 antibodies include Yervoy™ (ipilimumab or antibody10D1, described in PCT Publication WO 01/14424), tremelimumab (formerlyticilimumab, CP-675,206), or an anti-CTLA-4 antibody described in any ofthe following publications: WO 98/42752; WO 00/37504; U.S. Pat. No.6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) CancerRes. 58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed inWO2013/173223 may also be used.

Exemplary anti-LAG3 antibodies include antibodies comprising the CDRs orvariable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5,which are described in U.S. Patent Publication No. US2011/0150892,WO10/19570 and WO2014/008218. In one embodiment, an anti-LAG-3 antibodyis BMS-986016. Other art recognized anti-LAG-3 antibodies that can beused include IMP731 and IMP-321, described in US 2011/007023,WO08/132601, and WO09/44273.

In certain embodiments, the anti-OX40 antibody is used in combinationwith ipilimumab. In certain embodiments, the antibody has heavy andlight chain CDRs or variable regions of ipilimumab. Accordingly, in oneembodiment, the antibody comprises CDR1, CDR2, and CDR3 domains of theVH of ipilimumab having the sequence set forth in SEQ ID NO: 309, andCDR1, CDR2 and CDR3 domains of the VL of ipilimumab having the sequenceset forth in SEQ ID NO: 310. In certain embodiments, the antibodycomprises CDR1, CDR2 and CDR3 domains comprising the sequences set forthin SEQ ID NOs: 311-313, respectively, and CDR1, CDR2 and CDR3 domainscomprising the sequences set forth in SEQ ID NOs: 314-316, respectively.

In certain embodiments, the antibody comprises VH and/or VL regionscomprising the amino acid sequences set forth in SEQ ID NO: 309 and/orSEQ ID NO: 310, respectively. In certain embodiments, the antibody hasat least about 90%, e.g., at least about 90%, 95%, or 99% variableregion identity with SEQ ID NO: 309 or SEQ ID NO: 310.

Administration anti-OX40 antibodies and antagonists, e.g., antagonistantibodies, to one or more second target antigens such as LAG-3 and/orCTLA-4 and/or PD-1 and/or PD-L1 can enhance the immune response tocancerous cells in the patient. Cancers whose growth may be inhibitedusing anti-OX40 antibodies include cancers typically responsive toimmunotherapy and those that are not typically responsive toimmunotherapy. Representative examples of cancers for treatment with thecombination therapy of the instant disclosure include those cancerslisted herein.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. Furthermore, ifmore than one dose of the combination therapy is administeredsequentially, the order of the sequential administration can be reversedor kept in the same order at each time point of administration, andsequential administrations can be combined with concurrentadministrations, or any combination thereof. For example, the firstadministration of a combination of anti-OX40 antibody and anti-PD1antibody (and/or anti-CTLA-4 antibody and/or anti-PD-L1 antibody and/oranti-LAG-3 antibody) can be concurrent, the second administration can besequential with anti-PD1 antibody first and the anti-OX40 antibodysecond, and the third administration can be sequential with theanti-OX40 antibody first and anti-PD1 antibody second, etc. Anotherrepresentative dosing scheme involves a first administration that issequential with the anti-OX40 first and anti-PD1 antibody (and/oranti-CTLA-4 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3antibody) second, and subsequent administrations may be concurrent.

In certain embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of the anti-OX40 antibodyand an immuno-oncology agent. Exemplary immune-oncology agents includeCD137 (4-1BB) agonists (e.g., an agonistic CD137 antibody such asurelumab or PF-05082566 (WO12/32433)); GITR agonists (e.g., an agonisticanti-GITR antibody), CD40 agonists (e.g., an agonistic CD40 antibody);CD40 antagonists (e.g., an antagonistic CD40 antibody such aslucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4);CD27 agonists (e.g., an agonistic CD27 antibody such as varlilumab(CDX-1127)), MGA271 (to B7H3) (WO11/109400)); KIR antagonists (e.g.,lirilumab); IDO antagonists (e.g., INCB-024360 (WO2006/122150,WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620,WO09/1156652, WO11/56652, WO12/142237) or F001287); Toll-like receptoragonists (e.g., TLR2/4 agonists (e.g., Bacillus Calmette-Guerin); TLR7agonists (e.g., Hiltonol or Imiquimod); TLR7/8 agonists (e.g.,Resiquimod); or TLR9 agonists (e.g., CpG7909)); and TGF-β inhibitors(e.g., GC1008, LY2157299, TEW7197, or IMC-TR1).

In one embodiment, the anti-OX40 antibody is administered prior toadministration of a second agent, e.g., an immuno-oncology agent. Inanother embodiment, the anti-OX40 antibody is administered concurrentlywith the second agent, e.g., an immunology-oncology agent. In yetanother embodiment, the anti-OX40 antibody is administered afteradministration of the second agent. The administration of the two agentsmay start at times that are, e.g., 30 minutes, 60 minutes, 90 minutes,120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3days, 5 days, 7 days, or one or more weeks apart, or administration ofthe second agent may start, e.g., 30 minutes, 60 minutes, 90 minutes,120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3days, 5 days, 7 days, or one or more weeks after the first agent hasbeen administered.

In certain embodiments, the anti-OX40 antibody and a second agent, e.g.,an immuno-oncology agent, are administered simultaneously, e.g., areinfused simultaneously, e.g., over a period of 30 or 60 minutes, to apatient. The anti-OX40 antibody may be co-formulated with the secondagent, e.g., an immuno-oncology agent.

Optionally, the anti-OX40 antibody as sole immunotherapeutic agent, or acombination of the anti-OX40 antibody and one or more additionalimmunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 blockade), can be further combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below). A combination of the anti-OX40 antibody andone or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade) can also be further combined with standard cancertreatments. For example, a combination of the anti-OX40 antibody and oneor more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade) can be effectively combined with chemotherapeuticregimes. In these instances, the dose of other chemotherapeutic reagentadministered with the combination can be reduced (Mokyr et al. (1998)Cancer Research 58: 5301-5304). For example, such a combination mayinclude the anti-OX40 antibody with or without and an additionalantibody (e.g., anti-CTLA-4 antibodies and/or anti-PD-1 antibodiesand/or anti-PD-L1 antibodies and/or anti-LAG-3 antibodies), further incombination with decarbazine or interleukin-2 (IL-2) for the treatmentof melanoma. The scientific rationale behind combining an agonisticanti-OX40 antibody with CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3blockade with chemotherapy is that cell death, which is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy with acombination of the anti-OX40 antibody with or without and CTLA-4 and/orPD-1 and/or PD-L1 and/or LAG-3 blockade through cell death includeradiation, surgery, or hormone deprivation. Each of these protocolscreates a source of tumor antigen in the host. Angiogenesis inhibitorscan also be combined with a combination of the anti-OX40 antibody andCTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade. Inhibition ofangiogenesis leads to tumor cell death, which can be a source of tumorantigen fed into host antigen presentation pathways.

In certain embodiments, the anti-OX40 antibody can be used as the soleimmunotherapeutic agent, or a combination of the anti-OX40 antibody andCTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocking antibodies, canalso be used in combination with bispecific antibodies that target Fcαor Fcγ receptor-expressing effector cells to tumor cells (see, e.g.,U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can beused to target two separate antigens. The T cell arm of these responseswould be augmented by the use of a combination of the anti-OX40 antibodyand CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade.

In another example, the anti-OX40 antibody can be used as the soleimmunotherapeutic agent, or a combination of the anti-OX40 antibody andadditional immunostimulating agent, e.g., anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or LAG-3 agent (e.g.,antibody) can be used in conjunction with an anti-neoplastic antibody,such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva®(erlotinib), and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated by theimmunostimulating agent (e.g., OX40, CTLA-4, PD-1, PD-L1 or LAG-3 agent,e.g., antibody). In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) can include ananti-cancer agent (e.g., antibody) in combination with the anti-OX40antibody and optionally an additional immunostimulating agent, e.g.,anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent(e.g., antibody), concurrently or sequentially or any combinationthereof, which can potentiate an anti-tumor immune responses by thehost.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). Antibodies to each of these entities can befurther combined with the anti-OX40 antibody with or without anadditional immunostimulating agent, e.g., an anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, such as antibody,to counteract the effects of immunosuppressive agents and favoranti-tumor immune responses by the host.

Other agents (e.g., antibodies) that can be used to activate host immuneresponsiveness can be further used in combination with the anti-OX40antibody with or without an additional immunostimulating agent, such asanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibody. These include molecules on the surface of dendritic cells thatactivate DC function and antigen presentation. Anti-CD40 antibodies(Ridge et al., supra) can be used in conjunction with the anti-OX40antibody and optionally an additional immunostimulating agent, e.g., ananti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent,e.g., antibody. Other activating antibodies to T cell costimulatorymolecules Weinberg et al., supra, Melero et al. supra, Hutloff et al.,supra, may also provide for increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. Anti-OX40immunotherapy alone or combined with CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade can be used to increase the effectiveness of thedonor engrafted tumor specific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Er vivo activation inthe presence of the anti-OX40 antibody with or without an additionalimmunostimulating therapy, e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 antibodies can be expected to increase thefrequency and activity of the adoptively transferred T cells.

Provided herein are methods for altering an adverse event associatedwith the treatment of a hyperproliferative disease (e.g., cancer) withan immunostimulatory agent, comprising administering the anti-OX40antibody with or without an anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 agent (e.g., antibody), to a subject. Forexample, the methods described herein provide for a method of reducingthe incidence of immunostimulatory therapeutic antibody-induced colitisor diarrhea by administering a non-absorbable steroid to the patient. Asused herein, a “non-absorbable steroid” is a glucocorticoid thatexhibits extensive first pass metabolism such that, following metabolismin the liver, the bioavailability of the steroid is low, i.e., less thanabout 20%. In one embodiment, the non-absorbable steroid is budesonide.Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration.ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulationof budesonide developed to optimize drug delivery to the ileum andthroughout the colon. ENTOCORT EC® is approved in the U.S. for thetreatment of mild to moderate Crohn's disease involving the ileum and/orascending colon. The usual oral dosage of ENTOCORT EC® for the treatmentof Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in theintestines before being absorbed and retained in the gut mucosa. Once itpasses through the gut mucosa target tissue, ENTOCORT EC® is extensivelymetabolized by the cytochrome P450 system in the liver to metaboliteswith negligible glucocorticoid activity. Therefore, the bioavailabilityis low (about 10%). The low bioavailability of budesonide results in animproved therapeutic ratio compared to other glucocorticoids with lessextensive first-pass metabolism. Budesonide results in fewer adverseeffects, including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, the anti-OX40 antibody with or withoutCTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade (i.e., anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) inconjunction with a non-absorbable steroid can be further combined with asalicylate. Salicylates include 5-ASA agents such as, for example:sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine (DIPENTUM®,Pharmacia & UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals,Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals;PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

In accordance with the methods described herein, a salicylateadministered in combination with the anti-OX40 antibody with or withoutanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodiesand a non-absorbable steroid can include any overlapping or sequentialadministration of the salicylate and the non-absorbable steroid for thepurpose of decreasing the incidence of colitis induced by theimmunostimulatory antibodies. Thus, for example, methods for reducingthe incidence of colitis induced by the immunostimulatory antibodiesdescribed herein encompass administering a salicylate and anon-absorbable concurrently or sequentially (e.g., a salicylate isadministered 6 hours after a non-absorbable steroid), or any combinationthereof. Further, a salicylate and a non-absorbable steroid can beadministered by the same route (e.g., both are administered orally) orby different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-OX40 antibody and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies.

Anti-OX40 antibodies and combination antibody therapies described hereinmay also be used in conjunction with other well-known therapies that areselected for their particular usefulness against the indication beingtreated (e.g., cancer). Combinations with anti-OX40 antibodies may beused sequentially with known pharmaceutically acceptable agent(s).

For example, anti-OX40 antibodies and combination antibody therapiesdescribed herein can be used in combination (e.g., simultaneously orseparately) with an additional treatment, such as irradiation,chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU),cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, orcamptothecin+apo2l/TRAIL (a 6× combo)), one or more proteasomeinhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors(e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor(e.g., INCB24360, indoximod, NLG-919, or F001287), AT-101(R-(−)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1)antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g.,smac7, smac4, small molecule smac mimetic, synthetic smac peptides (seeFulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), orAEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20antibodies (e.g., rituximab), angiogenesis inhibitors (e.g.,bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g.,Avastin), synthetic triterpenoids (see Hyer et al., Cancer Research2005; 65:4799-808), c-FLIP (cellular FLICE-inhibitory protein)modulators (e.g., natural and synthetic ligands of PPARγ (peroxisomeproliferator-activated receptor γ), 5809354 or 5569100), kinaseinhibitors (e.g., Sorafenib), Trastuzumab, Cetuximab, Temsirolimus, mTORinhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK3βinhibitors, IAP inhibitors and/or genotoxic drugs.

Anti-OX40 antibodies and combination antibody therapies described hereincan further be used in combination with one or more anti-proliferativecytotoxic agents. Classes of compounds that may be used asanti-proliferative cytotoxic agents include, but are not limited to, thefollowing:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with anti-OX40antibodies, without limitation, taxanes, paclitaxel (paclitaxel iscommercially available as TAXOL™), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D,cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with the anti-OX40antibody, hormones and steroids (including synthetic analogs), such as17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, ZOLADEX™, can also be administered to the patient. Whenemploying the methods or compositions described herein, other agentsused in the modulation of tumor growth or metastasis in a clinicalsetting, such as antimimetics, can also be administered as desired.

In certain embodiments, the anti-OX40 antibody is administered incombination (concurrently or separately) with nivolumab to treat apatient with cancer, for example, colorectal or bladder cancer.

In certain embodiments, the anti-OX40 antibody is administered incombination (concurrently or separately) with ipilimumab to treat apatient with cancer, for example, ovarian, bladder, or prostate cancer.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto. The chemotherapeuticagent(s) and/or radiation therapy can be administered according totherapeutic protocols well known in the art. It will be apparent tothose skilled in the art that the administration of the chemotherapeuticagent(s) and/or radiation therapy can be varied depending on the diseasebeing treated and the known effects of the chemotherapeutic agent(s)and/or radiation therapy on that disease. Also, in accordance with theknowledge of the skilled clinician, the therapeutic protocols (e.g.,dosage amounts and times of administration) can be varied in view of theobserved effects of the administered therapeutic agents on the patient,and in view of the observed responses of the disease to the administeredtherapeutic agents.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents, and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

XVII. Kits and Unit Dosage Forms

Also provided herein are kits which include a pharmaceutical compositioncontaining an anti-OX40 antibody (e.g., OX40.21) and an anti-PD-1 (e.g.,nivolumab) or anti-CTLA-4 (ipilimumab) antibody, and apharmaceutically-acceptable carrier, in a therapeutically effectiveamount adapted for use in the preceding methods. The kits optionallyalso can include instructions, e.g., comprising administrationschedules, to allow a practitioner (e.g., a physician, nurse, orpatient) to administer the composition contained therein to administerthe composition to a patient having cancer (e.g., a solid tumor). Thekit also can include a syringe.

Optionally, the kits include multiple packages of the single-dosepharmaceutical compositions each containing an effective amount of theanti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody for a singleadministration in accordance with the methods provided above.Instruments or devices necessary for administering the pharmaceuticalcomposition(s) also may be included in the kits. For instance, a kit mayprovide one or more pre-filled syringes containing an amount of theanti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody.

In one embodiment, the present invention provides a kit for treating asolid tumor in a human patient, the kit comprising a dose of ananti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 318,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94, and instructions for usein the methods described herein. In certain embodiments, the kit furthercomprises (a) a dose of an anti-PD-1 antibody comprising CDR1, CDR2 andCDR3 domains of the heavy chain variable region having the sequence setforth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3 domains of the lightchain variable region having the sequence set forth in SEQ ID NO: 302,or (b) a dose of an anti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3domains of the heavy chain variable region having the sequence set forthin SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the light chainvariable region having the sequence set forth in SEQ ID NO: 310.

EMBODIMENTS

1. An isolated antibody, or antigen binding portion thereof, which bindsto human OX40 and exhibits the following properties:

(a) binds to membrane-bound human OX40;

(b) binds to cynomolgus OX40;

(c) binds to soluble human OX40;

(d) induces or enhances T cell activation;

(e) inhibits the binding of OX40 ligand to OX40;

(f) competes for binding to human OX40 with one or more of antibodies3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1,14A2-2, and 20C1.

2. The antibody of embodiment 1, wherein the antibody does not bind tomouse and/or rat OX40.3. The antibody, or antigen binding portion thereof, of embodiment 1 or2, wherein the antibody stimulates an anti-tumor immune response.4. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody stimulates anantigen-specific T cell response.5. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody increases IL-2 and/or IFN-γproduction in OX40-expressing T cells.6. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody increases T cellproliferation.7. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to Fc receptors.8. The antibody, or antigen binding portion thereof, of embodiment 7,wherein the antibody binds to one or more activating FcγRs.9. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to soluble human OX40with a K_(D) of about 1 nM or less, such as 0.5 nM or less or 0.1 nM orless, as measured by Biacore.10. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to membrane boundhuman OX40 with an EC₅₀ of 50 nM or less, such as 10 nM or less or 1 nMor less, as measured by FACS.11. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to membrane boundcynomolgus OX40 with an EC₅₀ of 50 nM or less, such as 10 nM or less or1 nM or less as measured by FACS.12. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody induces or enhances T cellactivation through multivalent cross-linking.13. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds the C1q component ofhuman complement.14. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody induces NK cell-mediatedlysis of activated CD4+ T cells.15. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody promotes macrophage-mediatedphagocytosis of OX40 expressing cells.16. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody inhibits regulatory Tcell-mediated suppression of CD4+ T cell proliferation.17. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to the sequenceDVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).18. The antibody, or antigen binding portion thereof, of any one ofembodiments 1-16, wherein the antibody binds to the sequenceDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40 (SEQID NO: 2).19. An isolated monoclonal antibody, or antigen binding portion thereof,which specifically binds to OX40 and comprises the three variable heavychain CDRs and the three variable light chain CDRs that are in thevariable heavy chain and variable light chain pairs selected from thegroup consisting of:

(a) SEQ ID NOs: 318 and 94;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 28 and 29;

(d) SEQ ID NOs: 28 and 30;

(e) SEQ ID NOs: 37 and 38;

(f) SEQ ID NOs: 48 and 49;

(g) SEQ ID NOs: 48 and 50;

(h) SEQ ID NOs: 57 and 58;

(i) SEQ ID NOs: 65 and 66;

(j) SEQ ID NOs: 73 and 74;

(k) SEQ ID NOs: 84 and 85;

(l) SEQ ID NOs: 84 and 86; and

(m) SEQ ID NOs: 93 and 94.

20. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40, comprising:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87, 317, and 89, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:11-13, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 14-16, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 22-24, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 25-27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:31-33, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 34-36, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 42-44, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 45-47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:51-53, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 54-56, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:59-61, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 62-64, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:67-69, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 70-72, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 78-80, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 81-83, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:87-89, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 90-92, respectively.

21. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40, comprising:

(a) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:87, 317, and 89, respectively, and/or light chain CDR1, CDR2, and CDR3sequences consisting of SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:11-13, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 14-16, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 22-24, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:19-21, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 25-27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:31-33, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 34-36, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 42-44, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:39-41, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 45-47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:51-53, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 54-56, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:59-61, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 62-64, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:67-69, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 70-72, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 78-80, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:75-77, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 81-83, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOs:87-89, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencesconsisting of SEQ ID NOs: 90-92, respectively.

22. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40 and comprises heavy and light chain variableregions, wherein the heavy chain variable region comprises an amino acidsequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 17, 28, 37, 48, 57,65, 73, 84, and 93.23. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40 and comprises heavy and light chain variableregions, wherein the light chain variable region comprises an amino acidsequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 18, 29, 30, 38, 49,50, 58, 66, 74, 85, 86, and 94.24. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40 and comprises heavy and light chain variable regionsequences at least 85% identical to the amino acid sequences selectedfrom the group consisting of:

(a) SEQ ID NOs: 318 and 94;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 28 and 29;

(d) SEQ ID NOs: 28 and 30;

(e) SEQ ID NOs: 37 and 38;

(f) SEQ ID NOs: 48 and 49;

(g) SEQ ID NOs: 48 and 50;

(h) SEQ ID NOs: 57 and 58;

(i) SEQ ID NOs: 65 and 66;

(j) SEQ ID NOs: 73 and 74;

(k) SEQ ID NOs: 84 and 85;

(l) SEQ ID NOs: 84 and 86; and

(m) SEQ ID NOs: 93 and 94.

25. The antibody, or antigen binding portion thereof, of embodiment 24,wherein the heavy and light chain variable regions comprise an aminoacid sequence at least 90% identical to the heavy and light chainvariable regions selected from the group consisting of (a)-(l) ofembodiment 24.26. The antibody, or antigen binding portion thereof, of embodiment 25,wherein the heavy and light chain variable region comprises an aminoacid sequence at least 95% identical to the heavy and light chainvariable regions selected from the group consisting of (a)-(l) ofembodiment 24.27. The antibody, or antigen binding portion thereof, of embodiment 26,wherein the heavy and light chain variable region comprises the heavyand light chain variable regions selected from the group consisting of(a)-(l) of embodiment 24.28. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to OX40 and comprises heavy chain and light chain sequencesat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequences selected from the group consisting of:

(a) SEQ ID NOs: 124 and 116, respectively;

(b) SEQ ID NOs: 95 and 96, respectively;

(c) SEQ ID NOs: 97 and 98, respectively;

(d) SEQ ID NOs: 99 and 100, respectively;

(e) SEQ ID NOs: 101 and 102, respectively;

(f) SEQ ID NOs: 103 and 104, respectively;

(g) SEQ ID NOs: 105 and 106, respectively;

(h) SEQ ID NOs: 107 and 108, respectively;

(i) SEQ ID NOs: 109 and 110, respectively;

(j) SEQ ID NOs: 111 and 112, respectively;

(k) SEQ ID NOs: 113 and 114, respectively;

(l) SEQ ID NOs: 115 and 116, respectively;

(m) SEQ ID NOs: 117 and 118, respectively;

(n) SEQ ID NOs: 119 and 120, respectively;

(o) SEQ ID NOs: 121 and 122, respectively;

(p) SEQ ID NOs: 123 and 116, respectively; and

(q) SEQ ID NOs: 125 and 116, respectively.

29. The antibody, or antigen binding portion thereof, of embodiment 28,wherein the heavy and light chains comprises the heavy and light chainsselected from the group consisting of (a)-(r) of embodiment 28.30. An isolated monoclonal antibody, or antigen binding portion thereof,which (a) binds to the same epitope on OX40 as the antibody ofembodiment 27, and/or (b) inhibits binding of the antibody of embodiment27 to OX40 on activated T cells by at least 95% as measured by FACS.31. The antibody, or antigen binding portion thereof, of any one ofembodiments 19-30, wherein the antibody binds to the sequenceDVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).32. The antibody, or antigen binding portion thereof, of any one ofembodiments 19-30, wherein the antibody binds to the sequenceDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40 (SEQID NO: 2).33. The antibody, or antigen binding portion thereof, of any one ofembodiments 19-31, wherein the antibody binds to both human andcynomolgus OX40.34. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody is selected from the groupconsisting of an IgG1, an IgG2, an IgG3, an IgG4, or a variant thereof.35. The antibody, or antigen binding portion thereof, of embodiment 34,wherein the antibody is an IgG1 antibody.36. The antibody of embodiment 35, wherein the antibody, or antigenbinding portion thereof, comprises an Fc having enhanced binding to anactivating FcγR.37. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein one or more methionine residues in theCDR regions are substituted for amino acid residues that do not undergooxidation.38. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody, or antigen binding portionthereof, is a human or humanized antibody.39. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody is not immunogenic, asassessed according to Example 21.40. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the amino acid sequence Asp-Gly, ifpresent in the heavy and/or light chain CDR sequences, is substitutedwith an amino acid sequence that does not undergo isomerization.41. The antibody, or antigen binding portion thereof, of embodiment 40,wherein the antibody comprises the heavy chain variable region CDR2sequence set forth in SEQ ID NO: 76, but wherein the Asp-Gly sequence isreplaced an amino acid sequence that does not undergo isomerization.42. The antibody of embodiment 41, wherein the Asp or Gly in the Asp-Glysequence is replaced with Ser.43. A bispecific molecule comprising the antibody of any one of thepreceding embodiments linked to a molecule having a second bindingspecificity.44. A nucleic acid encoding the heavy and/or light chain variable regionof the antibody, or antigen binding portion thereof, of any one ofembodiments 1-42.45. An expression vector comprising the nucleic acid molecule ofembodiment 44.46. A cell transformed with an expression vector of embodiment 45.47. An immunoconjugate comprising the antibody according to any one ofembodiments 1-42, linked to an agent.48. A composition comprising the antibody, or antigen binding portionthereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47, and a carrier.49. A kit comprising the antibody, or antigen binding portion thereof,or bispecific molecule, or immunoconjugate of any one of embodiments1-43 and 47 and instructions for use.50. A method of preparing an OX40 antibody, or antigen binding portionthereof, comprising expressing the antibody, or antigen binding portionthereof, in the cell of embodiment 46 and isolating the antibody, orantigen binding portion thereof, from the cell.51. A method of stimulating an antigen-specific T cell responsecomprising contacting the T cell with the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47 such that an antigen-specific T cell response isstimulated.52. A method of activating or co-stimulating an effector T cell,comprising contacting an effector T cell with an anti-OX40 antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47 and CD3, wherein the effector Tcell is activated or co-stimulated.53. A method of increasing IL-2 and/or IFN-γ production in a T cellcomprising contacting the T cell with an effective amount of theantibody, or antigen binding portion thereof, bispecific molecule orimmunoconjugate, of any one of embodiments 1-43 and 47.54. A method of increasing T cell proliferation comprising contactingthe cell with an effective amount of the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47.55. A method of increasing IL-2 and/or IFN-γ production in T cells in asubject comprising administering an effective amount of the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47, to increase IL-2 and/or IFN-γproduction from the T cells.56. A method of reducing or depleting the number of T regulatory cellsin a tumor of a subject in need thereof comprising administering aneffective amount of an antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-43and 47, wherein the antibody, or antigen binding portion thereof, haseffector or enhanced effector function, to reduce the number of Tregulatory cells in the tumor.57. A method of stimulating an immune response in a subject comprisingadministering the antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-43and 47 to the subject such that an immune response in the subject isstimulated.58. The method of embodiment 57, wherein the subject has a tumor and animmune response against the tumor is stimulated.59. A method for inhibiting the growth of tumor cells in a subjectcomprising administering to the subject the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47, such that growth of the tumor is inhibited.60. A method of treating cancer comprising administering to a subject inneed thereof a therapeutically effective amount of the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47, to treat the cancer.61. The method of embodiment 60, wherein the cancer is selected from thegroup consisting of: bladder cancer, breast cancer, uterine/cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer.62. The method of embodiment 60 or 61 wherein the cancer is a metastaticcancer, refractory cancer, or recurrent cancer.63. The method of any one of embodiments 56-62, further comprisingadministering one or more additional therapeutics.64. The method of embodiment 63, wherein the one or more additionaltherapeutics is an antibody or a small molecule.65. The method of embodiment 64, wherein the additional therapy is ananti-PD1 antibody, a LAG-3 antibody, a CTLA-4 antibody, a PD-L1antibody, or an anti-TGFβ antibody.66. A method of treating a solid tumor in a human subject, the methodcomprising administering to the subject an effective amount of ananti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 318,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94, wherein the methodcomprises at least one administration cycle, wherein the cycle is aperiod of two weeks, wherein for each of the at least one cycles, onedose of the anti-OX40 antibody is administered at a dose of 1 mg/kg bodyweight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about 1mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or 320 mg.67. A method of treating a solid tumor in a human subject, the methodcomprising administering to the subject an effective amount of each of:

(a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:318, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94,

(b) an anti-PD-1 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:301, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 302,

wherein the method comprises at least one administration cycle, whereinthe cycle is a period of two, three, or four weeks, wherein for each ofthe at least one cycles, one dose of the anti-OX40 antibody isadministered at a dose of 1 mg/kg body weight; a fixed dose of 20, 40,80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or a fixed doseof about 20, 40, 80, 160, or 320 mg, and one dose of the anti-PD-1antibody is administered at a dose of 240, 360, or 480 mg or a dose ofabout 240, 360, or 480 mg.

68. The method of embodiment 67, wherein the anti-OX40 antibody andanti-PD-1 antibody are administered at the following doses:

(a) 1 mg/kg body weight anti-OX40 antibody and 240 mg, 360 mg, or 480 mgof anti-PD-1 antibody;

(b) 20 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(c) 40 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(d) 80 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody;

(e) 160 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody; or

(f) 320 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg of anti-PD-1antibody.

69. A method of treating a solid tumor in a human subject, the methodcomprising administering to the subject an effective amount of each of:

(a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of theheavy chain variable region having the sequence set forth in SEQ ID NO:318, and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 94,

(b) an anti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3 domains ofthe heavy chain variable region having the sequence set forth in SEQ IDNO: 309, and CDR1, CDR2 and CDR3 domains of the light chain variableregion having the sequence set forth in SEQ ID NO: 310,

wherein the method comprises at least one administration cycle, whereinthe cycle is a period of three weeks, wherein for each of the at leastone cycles, one dose of the anti-OX40 antibody is administered at a doseof 1 mg/kg body weight; a fixed dose of 20, 40, 80, 160, or 320 mg; adose of about 1 mg/kg body weight; or a fixed dose of about 20, 40, 80,160, or 320 mg, and one dose of the anti-CTLA-4 antibody is administeredat a dose of 1 mg/kg or a dose of about 1 mg/kg,

wherein the anti-OX40 antibody is administered together with theanti-CTLA-4 antibody for at least one cycle, followed by anti-OX40antibody monotherapy for at least one cycle.

70. The method of embodiment 67, wherein the anti-OX40 antibody andanti-CTLA-4 antibody are administered at the following doses:

(a) 1 mg/kg body weight anti-OX40 antibody and 1 mg/kg anti-CTLA-4antibody;

(b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody; or

(f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody.

71. The method of any one of embodiments 66-70, wherein the anti-OX40antibody, or anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody,are formulated for intravenous administration.72. The method of any one of embodiments 67-71, wherein the anti-OX40and anti-PD-1 or anti-CTLA-4 antibody are formulated together.73. The method of any one of embodiments 67-71, wherein the anti-OX40and anti-PD-1 or anti-CTLA-4 antibody are formulated separately.74. The method of any one of embodiments 66-68, and 71-73, wherein thetreatment consists of up to 12 cycles.75. The method of any one of embodiments 69-73, wherein the treatmentconsists of 8 cycles.76. The method of embodiment 75, wherein the anti-OX40 antibody isadministered together with the anti-CTLA-4 antibody for the first 4cycles, followed by anti-OX40 antibody monotherapy for the last 4cycles.77. The method of any one of embodiments 66-76, wherein the anti-OX40antibody, or anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody,are administered on Day 1 of each cycle.78. The method of any one of embodiments 67-77, wherein the anti-OX40antibody is administered prior to administration of the anti-PD-1 oranti-CTLA-4 antibody.79. The method of embodiment 78, wherein the anti-OX40 antibody isadministered within about 30 minutes prior to administration of theanti-PD-1 or anti-CTLA-4 antibody.80. The method of any one of embodiments 67-77, wherein the anti-OX40antibody is administered after administration of the anti-PD-1 oranti-CTLA-4 antibody.81. The method of any one of embodiments 67-77, wherein the anti-OX40antibody is administered concurrently with the anti-PD-1 or anti-CTLA-4antibody.82. The method of any one of embodiments 66-81, wherein the treatmentproduces at least one therapeutic effect chosen from a reduction in sizeof a tumor, reduction in number of metastatic lesions over time,complete response, partial response, and stable disease.83. The method of any one of embodiments 66-82, wherein the solid tumoris associated with a cancer selected from the group consisting of:cervical cancer, bladder cancer, colorectal cancer, and ovarian cancer.84. The method of any one of embodiments 66-83, wherein the anti-OX40antibody comprises heavy chain and light chain variable region CDRscomprising the amino acid sequences set forth in SEQ ID NOs: 87, 317 and89, and 90-92, respectively.85. The method of any one of embodiments 66-84, wherein the anti-OX40antibody comprises heavy and light chain variable region sequences setforth in SEQ ID NOs: 318 and 94, respectively.86. The method of any one of embodiments 66-85, wherein the anti-OX40antibody comprises heavy and light chain sequences set forth in SEQ IDNOs: 124 and 116, respectively.87. The method of any one of embodiments 67, 68, and 71-86, wherein theanti-PD-1 antibody comprises heavy chain and light chain variable regionCDRs comprising the amino acid sequences set forth in SEQ ID NOs:303-305 and 306-308, respectively.88. The method of any one of embodiments 67, 68, and 71-87, wherein theanti-PD-1 antibody comprises heavy and light chain variable regionsequences set forth in SEQ ID NOs: 301 and 302, respectively.89. The method of any one of embodiments 69-86, wherein the anti-CTLA-4antibody comprises heavy chain and light chain variable region CDRscomprising the amino acid sequences set forth in SEQ ID NOs: 311-313 and314-316, respectively.90. The method of any one of embodiments 69-86 and 89, wherein theanti-CTLA-4 antibody comprises heavy and light chain variable regionsequences set forth in SEQ ID NOs: 309 and 310, respectively.91. A kit for treating a solid tumor in a human subject, the kitcomprising a dose of an anti-OX40 antibody comprising CDR1, CDR2 andCDR3 domains of the heavy chain variable region having the sequence setforth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the lightchain variable region having the sequence set forth in SEQ ID NO: 94,and instructions for use.92. The kit of embodiment 91, further comprising (a) a dose of ananti-PD-1 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 301,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 302, or (b) a dose of ananti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3 domains of the heavychain variable region having the sequence set forth in SEQ ID NO: 309,and CDR1, CDR2 and CDR3 domains of the light chain variable regionhaving the sequence set forth in SEQ ID NO: 310.93. A method of detecting the presence of OX40 in a sample comprisingcontacting the sample with the antibody, or antigen binding portionthereof, of any one of embodiments 1-38, under conditions that allow forformation of a complex between the antibody, or antigen binding.94. An antibody which binds to OX40 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 282-296.95. The antibody of embodiment 94, wherein the antibody comprises aheavy chain consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 282-296.96. An isolated monoclonal antibody which binds to OX40, comprisingheavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 87,317, and 89, respectively, and light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 90-92, respectively.97. An isolated monoclonal antibody which binds to OX40, comprisingheavy and light chain variable regions comprising the amino acidsequences of SEQ ID NOs: 318 and 94, respectively.98. An isolated monoclonal antibody which binds to OX40, comprisingheavy and light chains comprising the amino acid sequences of SEQ IDNOs: 124 and 116, respectively.99. An isolated monoclonal antibody which binds to OX40, wherein theantibody binds to all or a portion of the sequence DVVSSKPCKPCTWCNLR(SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).100. A composition comprising an isolated monoclonal antibody accordingto any one of embodiments 96-99 and a carrier.101. A nucleic acid encoding the heavy and/or light chain variableregion of the antibody of embodiment 96 or 97, or the heavy and/or lightchain of embodiment 98.102. A kit comprising the antibody of any one of embodiments 96-99.103. A method of stimulating an antigen-specific T cell responsecomprising contacting the T cell with an antibody according to any oneof embodiments 96-99.104. A method of treating cancer comprising administering to a subjectin need thereof a therapeutically effective amount of an antibodyaccording to any one of embodiments 96-99, to treat the cancer.105. The method of embodiment 104, wherein the cancer or solid tumor isselected from the group consisting of: cervical cancer, bladder cancer,colorectal cancer, ovarian cancer, non-small cell lung cancer, andsquamous cell carcinoma of the head and neck.

EXAMPLES Example 1: Generation of Anti-OX40 Antibodies

Human anti-OX40 monoclonal antibodies were generated in Hco7, Hco12,Hco17, and Hco38 strains of HuMAb® transgenic mice (“HuMAb” is a TradeMark of Medarex, Inc., Princeton, N.J.) and KM mice (the KM Mouse®strain contains the SC20 transchromosome as described in PCT PublicationWO 02/43478) using recombinant hexahistidine-OX40 antigen.

A total of 52 mice, including 5 genotypes of transgenic mice (KM, Hco7,Hco12, Hco17, and Hco38), were immunized with different immunizationstrategies. The immunogen was huOX40-6×his prepared in-house and used at2.0 mg/mL for a total dose of 20 μg per mouse. Routes of administrationincluded: base of tail injection, Hock immunization, intraperitoneal(ip) and subcutaneous (sc) injection, and adjuvant (Ribi, Cat #S6322,Sigma). 27 fusions from 30 mice were performed and screened. 541 ELISAantigen positive antibodies were identified from these 27 fusions, andfurther characterization led to the isolation of antibodies ofparticular interest, including the antibodies designated as 3F4, 14B6-1,14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3 (also referred to asOX40.17), 14A2-1, 14A2-2, and 20C1. Their variable region amino acidsequences and isotype are set forth in FIGS. 1A, 1B, 2A, 2B, 2C, 3A, 3B,4A, 4B, 4C, 5A, 5B, 6A, 6B, 7A, 7B, 8A, 8B, 8C, 9A, and 9B. The heavyand light chain variable regions of 3F4 consist of amino acid sequencesSEQ ID NOs: 17 and 18. The heavy and light chain variable regions of14B6-1 consist of amino acid sequences SEQ ID NOs: 28 and 29. The heavyand light chain variable regions of 14B6-2 consist of amino acidsequences SEQ ID NOs: 28 and 30. The heavy and light chain variableregions of 23H3 consist of amino acid sequences SEQ ID NOs: 37 and 38.The heavy and light chain variable regions of 6E1-1 consist of aminoacid sequences SEQ ID NOs: 48 and 49. The heavy and light chain variableregions of 6E1-2 consist of amino acid sequences SEQ ID NOs: 48 and 50.The heavy and light chain variable regions of 18E9 consist of amino acidsequences SEQ ID NOs: 57 and 58. The heavy and light chain variableregions of 8B11 consist of amino acid sequences SEQ ID NOs: 65 and 66.The heavy and light chain variable regions of 20B3 consist of amino acidsequences SEQ ID NOs: 73 and 74. The heavy and light chain variableregions of 14A2-1 consist of amino acid sequences SEQ ID NOs: 84 and 85.The heavy and light chain variable regions of 14A2-2 consist of aminoacid sequences SEQ ID NOs: 84 and 86. The heavy and light chain variableregions of 20C1 consist of amino acid sequences SEQ ID NOs: 93 and 94.

cDNA sequencing identified one heavy and one light chain for each of theantibodies 3F4, 23H3, 18E9, 8B11, 20B3 (also referred to as OX40.17) and20C1, and one heavy chain and two light chains (light chain 1 or “L1”and light chain 2 or “L2”) for each of the antibodies 14B6, 14A2 and6E1. By protein analysis, a single light chain was identified forantibodies 14B6, 6E1 and 14A2, and N-terminal sequencing and molecularweight determination indicated that it was light chain L1 for 14B6 and14A2 and light chain L2 for 6E1. Antibodies 14B6-1 and 14B6-2 correspondto antibody 14B6 with a light chain L1 and L2, respectively. Antibodies14A2-1 and 14A2-2 correspond to antibody 14A2 with a light chain L1 andL2, respectively. Antibodies 6E1-1 and 6E1-2 correspond to antibody 6E1with a light chain L1 and L2, respectively. The amino acid andnucleotide sequences of each of the light chains of the 3 antibodies areprovided in Table 23.

For some of the antibodies above, substitutions in the parental antibodywere made in HCDR2 in order to remove the presence of an isomerizationsite (DG), and framework substitutions (due to derivation from the DP44germline) were introduced to make the framework more like a commonlyexpressed antibody. For 20C1-based antibodies, three additional unusualframework residues were reverted to germline (A2V, D24G, and G82bS). TheG82bS framework reversion also eliminates a deamidation site (NG). Asummary of the various substitutions introduced into the parental hybridclone sequences is provided in Table 6.

TABLE 6 Parental hybridoma Variable region Name clone Isotypesubstitutions OX40.6 23H3 g1f Anti-OX40 23H3 with VH- H13Q/M87T OX40.723H3 g1f Anti-OX40 23H3 with VH- M87T/M95Y OX40.8 14A2 g1f Anti-OX4014A2 with VH- G103W OX40.9 14A2 g1f Anti-OX40 14A2 with VH- M97Y/G103WOX40.10 14A2 g1f Anti-OX40 14A2 with VH- D53S OX40.11 14A2 g1f Anti-OX4014A2 with VH- G54S OX40.12 14A2 g1f Anti-OX40 14A2 with VH- D53S/G103WOX40.13 14A2 g1f Anti-OX40 14A2 with VH- G54S/G103W OX40.14 14A2 g1fAnti-OX40 14A2 with VH- D53S/M97Y/G103W OX40.15 14A2 g1f Anti-OX40 14A2with VH- G54S/M97Y/G103W OX40.16 20C1 g1f Anti-OX40 20C1 with VH-A2V/H13Q/D24G/M87T/G82bS OX40.17 20B3 g1f Anti-OX40 20B3 (nosubstitutions) OX40.18 3F4 g1f Anti-OX40 3F4 with VH-N27Y/N72D/P102YOX40.19 14A2 g1f Anti-OX40 14A2 with VH-M97L/G103W OX40.20 20C1 g1fAnti-OX40 20C1 with VH- A2V/H13Q/D24G/D54S/M87T/G82bS OX40.21 20C1 g1fAnti-OX40 20C1 with VH- A2V/H13Q/D24G/G55A/M87T/G82bS OX40.22 20C1 g1fAnti-OX40 20C1 with VH- A2V/H13Q/D24G/ D54S/G55T/M87T/G82bS *Dependingon the germline, there may be a D53 and G54 or a D54 and G55 present, asa potential isomerization site

Example 2: Binding of Anti-OX40 Antibodies to Activated Primary Human TCells

The human monoclonal anti-OX40 antibodies generated in Example 1 weretested for the ability to bind to activated primary human T cells.

Cells were activated for several days before the binding assay in orderto induce OX40 expression. Briefly, PBMCs were cultured for three orfour days with magnetic beads coated with anti-human CD3 plus anti-humanCD28, in the presence of recombinant human IL-2. On the day of theassay, the beads were removed and the cells stained with a titration ofeach anti-OX40 antibody. Bound antibodies were detected with afluorescently conjugated anti-human IgG polyclonal secondary antibody,and the cells were co-stained for CD4 and CD25 to detect activated CD4 Tcells. The fluorescence intensity of the staining was measured using aFACSCanto II flow cytometer (Becton Dickinson). The geometric meanfluorescence intensity (GMFI) or median fluorescence intensity (MedFI)of anti-OX40 antibody staining was calculated for the CD4+CD25+population (FACSDiva software). EC₅₀s for antibody binding werecalculated using GraphPad Prism software.

As shown in FIG. 11A, the anti-OX40 antibodies bound to activatedprimary human T cells with subnanomolar EC₅₀s. Notably, OX40.5 showedlowest binding of the anti-OX40 antibodies tested. The same experimentwas performed with anti-OX40 antibodies with variable regionsubstitutions. Initial experiments were carried out using antibodies inthe form of supernatants from cultures of host cells transfected withrecombinant antibody expression vectors. As shown in FIG. 11B, certainsubstitutions caused a significant loss of binding, namely forantibodies OX40.7, OX40.9, OX40.14 and OX40.15. A set of antibodies withvariable region substitutions was analyzed further using purifiedantibody material.

As shown in FIGS. 11C and 11D, all tested antibodies bound withsubnanomolar EC50s to OX40, except for OX40.18, which showed lowerbinding than the 3F4 hybridoma parent clone. OX40.5 showed the lowestbinding among the panel of antibodies tested in FIG. 11B, whereas OX40.1showed the lowest binding among the panel of antibodies tested in FIG.11C. A summary of the EC₅₀ values is presented in Table 7 below.

TABLE 7 EC₅₀s for binding of OX40 antibodies to activated human primaryT cells. Human T Cell Binding EC50 (nM) Name (mean ± SD) n 3F4 0.19 ±0.15 3 8B11 0.14 ± 0.07 2 18E9 0.22 ± 0.12 2 20B3 0.34 ± 0.23 3 20C10.10 ± 0.06 3 23H3 0.15 ± 0.09 3 6E1 0.97 ± 0.05 2 14A2 0.33 ± 0.30 314B6 0.18 ± 0.16 2 OX40.6 0.13 ± 0.06 7 OX40.8 0.14 ± 0.07 7 OX40.160.06 ± 0.03 5 OX40.17 0.26 ± 0.17 3 OX40.18 3.15 ± 3.9  2 OX40.21 0.07 ±0.02 5 OX40.1 0.35 ± 0.09 2 OX40.4 0.36 ± 0.06 2 OX40.5 3.20 ± 0.00 2

Example 3: Binding of Anti-OX-40 Antibodies to Activated PrimaryCynomolgus Macaque T Cells

The human monoclonal anti-OX-40 antibodies that were tested for bindingto activated primary human T cells in Example 1 were tested for theability to bind activated primary cynomolgus macaque T cells.

Briefly, cells were activated for several days before the binding assayin order to induce OX40 expression. Total leukocytes were isolated fromcynomolgus macaque peripheral blood by lysis of red blood cells using anammonium chloride buffer. The leukocytes were then cultured for four tofive days with in flasks pre-coated with anti-human CD3 plus anti-humanCD28 antibodies that cross-react with cynomolgus macaque, in thepresence of recombinant human IL-2, in order to expand and activate Tcells. On the day of the assay, the cells were harvested and stainedwith a titration of each anti-OX40 antibody. Bound antibodies weredetected with a fluorescently conjugated anti-human IgG polyclonalsecondary antibody, and the cells were co-stained for CD4 and CD25 todetect activated CD4 T cells. The fluorescence intensity of the stainingwas measured using a FACSCanto II flow cytometer (Becton Dickinson). Thegeometric mean fluorescence intensity (GMFI) or median fluorescenceintensity (MedFI) of anti-OX40 antibody staining was calculated for theCD4+CD25+ population (FACSDiva software). Dose-response curves weregenerated and EC50s for antibody binding were calculated using GraphPadPrism software.

As shown in FIGS. 12A and 12B, the anti-OX40 antibodies tested boundwith high potency to activated cynomolgus macaque CD4 T cells, withEC₅₀s ranging from 0.068 nM (20C1) to 1.4 nM (20B3). 18E9 and 20B3 boundwith EC₅₀s between 1 and 1.5 nM, while the remaining antibodies boundwith EC₅₀s below 1 nM. OX40.1 showed the lowest binding among theanti-OX40 antibodies tested in FIG. 12A, and OX40.5 showed the lowestbinding among the anti-OX40 antibodies tested in FIG. 12B. The sameexperiment was performed with anti-OX40 antibodies with variable regionsubstitutions. As shown in FIG. 12C, OX40.6, OX40.8 and OX40.21antibodies showed the highest potency of binding, with EC₅₀s of 0.12 nMor lower. OX40.1 showed much lower binding to cynomolgous macaque CD4 Tcells. No binding of the OX40.21 antibody was detected on activatedmouse or rat CD4+ T cells. A summary of the EC₅₀ values is presented inTable 8 below.

TABLE 8 Cyno T Cell Binding EC50 (nM) Name (mean ± SD) n 3F4 0.37 ± 0.134 8B11 0.20 ± 0.08 4 18E9 41.70 ± 37.78 4 20B3 1.18 ± 0.27 4 20C1 * 0.17± 0.07 4 23H3 0.18 ± 0.10 4 6E1 0.77 ± 0.14 4 14A2 0.27 ± 0.02 4 14B60.31 1 OX40.6 0.11 ±0.01 5 OX40.8 0.10 ± 0.02 4 OX40.16 0.06 ± 0.01 3OX40.17 0.52 ± 0.12 3 OX40.18 OX40.21 0.07 ± 0.01 5 OX40.1 23.40 ± 37.754 OX40.4 1.3  1 OX40.5 ~2.2e+012 1

Example 4: Scatchard Analysis of Binding of Anti-OX40 Antibodies toActivated Primary T Cells and Cells Overexpressing Human and CynomolgusMonkey OX40

The binding of OX40.21 (IgG1 isotype) to activated human T cells wasfurther assessed using Scatchard analysis. Briefly, OX40.21 wasradioiodinated with ¹²⁵I-Na (1 mCi; PerkinElmer Catalog NEZ033H001 MC)using IODO-GEN® solid phase iodination reagent(1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; Pierce Catalog 28601).Activated human CD4+ T cells were isolated from peripheral bloodmononuclear cells (PBMC), Donor W-326470, purchased from the StanfordBlood Bank. CD4+ T cells were isolated by negative selection(RosetteSep™ Human CD4+ T cell enrichment cocktail, StemCellTechnologies Catalog 15062) and frozen. The isolated CD4+ T cells wereactivated for four days before the binding assay in order to induce OX40expression, as follows. Thawed cells were cultured for four days withmagnetic beads coated with anti-human CD3 plus anti-human CD28 (humanT-Expander CD3/CD28 Dynabeads, Invitrogen Catalog 111.41D), at a 1:1bead cell ratio, in the presence of 200 IU/mL recombinant human IL-2(Peprotech Catalog 200-02).

Radioiodinated OX40.21 IgG1 binding to activated human T cells wasdemonstrated by incubating activated human T cells with a titration of¹²⁵I-OX40.21 IgG1. Nonspecific binding was determined by binding in thepresence of a titration of a 100 fold molar excess of unlabeled antibodyand was subtracted from total CPM to calculate specific binding. Alinear standard curve of ¹²⁵I-OX40.21 IgG1 concentration versus CPM wasused to extrapolate specific activity, maximal nM bound ¹²⁵I-OX40.21IgG1 and thereby calculate receptor number per cell.

As shown in Table 9 and FIG. 13A, saturable binding of OX40.21 IgG1 wasobserved on activated human T cells endogenously expressing OX40, with aK_(D) of 0.05 nM for each of two T cell donors.

TABLE 9 Binding of ¹²⁵I-OX40.21 to Activated Human T Cells Total BindingNon-Specific Binding Specific Binding Ab Conc. ¹²⁵I-labeled Antibody¹²⁵I-labeled Antibody ¹²⁵I-labeled Antibody (nM) (nM) (nM) (nM) 100.0317 0.0292 0.0120 0.0117 0.0197 0.0175 5 0.0283 0.0275 0.0073 0.00720.0210 0.0204 2.5 0.0230 0.0256 0.0036 0.0030 0.0195 0.0226 1.25 0.02050.0221 0.0029 0.0025 0.0176 0.0196 0.625 0.0197 0.0223 0.0014 0.00170.0183 0.0207 0.3125 0.0197 0.0229 0.0013 0.0012 0.0184 0.0217 0.156250.0177 0.0201 0.0012 0.0011 0.0165 0.0190 0.078125 0.0140 0.0152 0.00100.0011 0.0129 0.0141 0.039063 0.0083 0.0091 0.0007 0.0010 0.0076 0.00810.019531 0.0057 0.0057 0.0009 0.0010 0.0049 0.0047 0.009766 0.00240.0030 0.0007 0.0008 0.0017 0.0022

The same assay was performed using HEK293 cells overexpressing humanOX40 (“hOX40-293”). Briefly, radioiodinated OX40.21 binding tooverexpressed human OX40 was demonstrated by incubating hOX40-293 cellswith a titration of ¹²⁵I-OX40.21. Nonspecific binding was determined bybinding in the presence of a titration of a 100 fold molar excess ofunlabeled antibody and was subtracted from total CPM to calculatespecific binding. A linear standard curve of ¹²⁵I-OX40.21 concentrationversus CPM was used to extrapolate maximal nM bound ¹²⁵I-OX40.21 andthereby calculate receptor numbers per cell. As shown in FIG. 13B andTable 10, saturable binding of OX40.21 IgG1 was observed for binding toOX40 expressed on hOX40-293 cells. The average K_(D) for binding fromtwo test conditions using different numbers of hOX40-293 cells persample was 0.22 nM.

TABLE 10 OX40.21 Binding to hOX40-293 Cells Total Binding Non-SpecificBinding Specific Binding Ab Conc. ¹²⁵I-labeled Antibody ¹²⁵I-labeledAntibody ¹²⁵I-labeled Antibody (nM) (nM) (nM) (nM) 10 0.1866 0.17120.0129 0.0137 0.1737 0.1575 5 0.1800 0.1710 0.0080 0.0099 0.1720 0.16112.5 0.1799 0.1643 0.0065 0.0065 0.1734 0.1578 1.25 0.1722 0.1628 0.00570.0054 0.1665 0.1574 0.625 0.1583 0.1436 0.0067 0.0048 0.1515 0.13880.3125 0.0986 0.0936 0.0038 0.0044 0.0948 0.0891 0.15625 0.0624 0.05010.0035 0.0048 0.0589 0.0453 0.078125 0.0351 0.0289 0.0035 0.0033 0.03160.0255 0.039063 0.0211 0.0162 0.0038 0.0027 0.0173 0.0136 0.0195310.0117 0.0091 0.0029 0.0028 0.0088 0.0063 0.009766 0.0075 0.0056 0.00270.0028 0.0048 0.0028

The same assay was performed using CHO cells overexpressing cynomolgusmonkey OX40 (“cynoOX40-CHO”). Briefly, radioiodinated OX40.21 binding tocynomologus OX40 was demonstrated by incubating cynoOX40-CHO cells witha titration of ¹²⁵I-OX40.21. Nonspecific binding was determined bybinding in the presence of a titration of a 100 fold molar excess ofunlabeled antibody and was subtracted from total CPM to calculatespecific binding. A linear standard curve of ¹²⁵I-OX40.21 concentrationversus CPM was used to extrapolate maximal nM bound ¹²⁵I-OX40.21 andthereby calculate receptor numbers per cell. As shown in FIG. 13C andTable 11, saturable binding of OX40.21 IgG1 was observed for binding tocynomologus OX40 expressed on cynoOX40-CHO cells. The average K_(D) forbinding from two test conditions using different numbers of cells persample was 0.63 nM.

TABLE 11 OX40.21 Binding to cynoOX40-CHO Cells Total BindingNon-Specific Binding Specific Binding Ab Conc. ¹²⁵I-labeled antibody¹²⁵I-labeled antibody ¹²⁵I-labeled antibody (nM) (nM) (nM) (nM) 200.1781 0.1814 0.0266 0.0414 0.1515 0.1400 10 0.1768 0.1651 0.0161 0.01970.1607 0.1454 5 0.1629 0.1820 0.0109 0.0171 0.1520 0.1649 2.5 0.16650.1659 0.0080 0.0092 0.1586 0.1567 1.25 0.1197 0.1839 0.0079 0.00840.1117 0.1755 0.625 0.0892 0.1197 0.0060 0.0074 0.0832 0.1123 0.31250.0630 0.0754 0.0057 0.0053 0.0573 0.0701 0.15625 0.0318 0.0437 0.00490.0050 0.0269 0.0386 0.078125 0.0158 0.0212 0.0030 0.0034 0.0128 0.01790.039063 0.0082 0.0110 0.0027 0.0029 0.0055 0.0082 0.019531 0.00580.0058 0.0026 0.0023 0.0032 0.0035 0.009766 0.0030 0.0032 0.0022 0.00210.0009 0.0011

Example 5: Specific Binding of Anti-OX40 Antibodies to Lymphocytes

The specificity of various OX40 antibodies was tested on a panel of 22normal human tissue types, including spleen, tonsil, thymus, cerebrum,cerebellum, heart, liver, lung, kidney, pancreas, pituitary, peripheralnerves, stomach, colon, small intestine, thyroid, skin, skeletal muscle,prostate, uterus, testes, and placenta by immunohistochemistry.

Fresh, frozen and/or OCT-embedded human tissues were purchased frommultiple commercial tissue networks/vendors (Asterand Inc. Detroit,Mich.; Cooperative Human Tissue Network, Philadelphia, Pa.; ProteoGenexInc, Culver City, Calif.). To detect tissue binding, a series ofanti-OX40 antibodies (OX40.6-FITC, OX40.8-FITC, 6E1-FITC, OX40.16-FITC,OX40.17-FITC, OX40.20-FITC, and OX40.21-FITC) were fluoresceinated andapplied to acetone fixed cryostat sections, followed by an anti-FITCbridging antibody and visualization by the EnVision+ System. Anonspecific fluoresceinated human IgG1 was used as isotype controlantibody. HT1080 cells stably expressing human OX40 (HT1080/huOX40) andhyperplasic human tonsil tissue sections were used as positive controlcells and tissues. To determine if FITC conjugation has any impact onbinding properties, both FITC-conjugated and un-conjugated anti-OX40antibodies were compared in HT1080/huOX40 cells using anti-huIgG asbridging antibody. Stained slides were evaluated under a lightmicroscope.

Initial tests revealed that both un-conjugated and FITC-conjugatedanti-OX40 antibodies specifically stained the cytoplasm and membrane ofhuman OX40 transfected cells but not parent HT1080 cells. There was nodifference between unconjugated and FITC-conjugated anti-OX40antibodies. These results suggest that the antibodies were suitable forimmunohistochemistry analyses, and that FITC conjugation has no impacton tissue binding properties.

All anti-OX40 antibodies tested exhibited positive staining in a smallsubset, either as scattered or small clusters, of mononuclear cells(MNC) in lymphoid tissues (tonsil, spleen, and thymus) and lymphoid-richtissues (colon, stomach, and small intestine), as well as a fewscattered MNC in multiple tissues (lung, skin, and thyroid). Based onmorphology, these positive cells are primarily lymphocytes.

In addition to staining a subset of lymphocytes, the OX40.6 antibody, aligand blocker, displayed strong staining in subsets ofendothelium/subendothelial matrix and interstitial elements, more oftenassociated with small arteries and adventitia of vessel and itssurrounding connective tissues, in virtually all tissues examined (FIG.14A), as well as specialized interstitial tissue elements such assheath-like interstitium surrounding the seminiferous tubule in thetestis. The OX40.8 antibody, a ligand non-blocker, positively labeledmyofilament-like structures in cardiac muscles of the heart (FIG. 14A)and mesangial-like cells in glomerulus of the kidney. Staining withanother ligand non-blocker, i.e., the 6E1 antibody, also revealedstaining in cardiac muscle cells, as well as in neurons and neuropils ofcerebrum and cerebellum and a subset of tubule epithelial cells inkidney. In general, the staining of non-lymphocytes was detected onlywhen the antibodies were used at relatively high concentrations (3 or 5μg/ml), but not at lower concentrations (1 μg/ml), suggesting lowaffinity binding or potential off-target binding.

Further testing of other ligand blocking antibodies, OX40.16 (FIG. 14A)and OX40.17, revealed clean staining of a subset of lymphocyte, with nospecific staining of other tissue elements for all tissues examined. TheOX40.21 antibody, a variant of the OX40.16 antibody, had a similarbinding pattern as the OX40.16 antibody (FIG. 14B). Immunohistochemistryin a similar panel of normal cynomolgus tissues revealed very similarstaining pattern to human, demonstrating the utility of cynomolgusmonkey as a relevant preclinical species.

Example 6: Expression of OX40 in Cancers

FFPE (formalin fixed paraffin embedded) tumor tissue samples werepurchased from commercial tissue venders (n=12-20 for each tumor type).To detect binding to tissues, an automated IHC assay with a commercialanti-human OX40 antibody was developed using the Leica BondRX platform.Briefly, heat-induced antigen retrieval (HIER) was performed in pH9 ER2buffer (Leica) for 20 min at 95° C. The mouse anti-human OX40 monoclonalantibody clone ACT35 (BD Pharmingen) was incubated at 5 μg/ml for 60minutes, followed by Novolink Max polymer (Leica) for 30 minutes.Finally, slides were reacted with DAB substrate-chromogen solution for 6minutes, counterstained with Mayer's hematoxylin, dehydrated, cleared,and coverslipped with Permount. Dako protein block was used as diluentfor the primary antibody.

To profile TILs, commercially available anti-CD3 (T cell marker) andanti-FoxP3 (Treg marker) monoclonal antibodies were used to stainadjacent sections. Commercial mouse IgG1 was used as a negative controland hyperplasic human tonsil tissue was used as a positive control.After immunostaining, slides were manually evaluated and scored under alight microscope.

In the four tumor types examined, CD3+ TILs were present in all samplesexamined, with the amount of TILs varying across samples and thedistribution within the same tissue heterogeneous. In some cases, TILswere more heavily distributed in the tumor and host interface, asexpected. Most TILs were localized in the tumor stroma in the vastmajority tissue samples. However, they were readily found inintratumoral nests in many cases. Positive OX40 staining was observed ina small fraction of TILs and primarily distributed in the tumor stroma.In general, the abundance of OX40+ TILs was in proportion to that ofCD3+ TILs. Among the four tumor types examined, OX40+ TILs were moreabundant in HCC and CRC (FIGS. 15A-15C).

Example 7: Human Monoclonal Anti-OX40 Antibodies that Block the Bindingof OX-40L to OX-40

Several anti-OX40 antibodies were tested for their ability to block thebinding of recombinant soluble OX40L to human OX40-transfected 293cells. Briefly, 293 cells stably transfected with human OX40 were firstpre-incubated with varying concentrations of anti-OX40 antibodies. Afixed concentration (0.2 μg/mL) of recombinant soluble his-tagged humanOX40L (OX40L-His, R & D Systems) was then added and the samplesincubated further. After washing the cells, bound OX40L-His was detectedusing an in-house APC-labeled anti-His tag antibody. The fluorescenceintensity of the staining was measured using a FACSCanto II flowcytometer (Becton Dickinson). The geometric mean fluorescence intensity(GMFI) of APC-anti-His tag antibody/OX40L-His staining for the cellpopulation was calculated (FACSDiva software). Dose-response curves weregenerated and EC50s for antibody blocking of OX40L binding werecalculated using GraphPad Prism software; the EC50s are shown in Table12.

TABLE 12 EC50 values for blocking of OX40L/OX40 interaction as measuredby FACS. Antibody Clone EC50 (nM) 14B6.C5.C8 1.0  3F4.G11.D2 0.548B11.H9.C1 0.45 18E9.G5.H4 0.48 20B3.G12.A2 0.93 20C1.F2.D1 0.346E1.A12.A2 no blocking 23H3.C6 0.38 OX40.4 no blocking OX40.5 ~1.2e+013

As shown in FIG. 16 , most of the anti-OX40 antibodies tested fullyblocked the binding of soluble human OX40L to human OX40 on the surfaceof transfected cells, with the exception of 6E1, OX40.4, and OX40.5. Theincomplete blocking by OX40.5 may be due to a lower potency of bindingto human OX40 than the other antibodies tested or binding to anoverlapping but different epitope. In contrast, 6E1 and OX40.4 did notblock the binding of human OX-40L to OX40. This lack of blocking islikely due to binding to a different epitope than the remainingantibodies.

Example 8: Antibody Competition/Binning

Antibody binning experiments were carried out as follows. One or moreanti-OX40 antibodies were coated directly onto a Biacore CM5 chip usingamine coupling chemistry. Anti-OX40 antibodies, serially diluted (1:3)from a starting concentration of 60 μg/mL, were incubated with 20 nM ofOX40-6×-His antigen for at least 1 hour. The incubated complex wasflowed over the antibody coupled surfaces and observed forcross-blocking. The exercise was repeated with several antibodies on thesurface to create the epitope map based on mutual cross-blocking of allthe antibodies. OX40L was also coated on the surface to identify and binthe antibodies that were able block OX40-OX40L interaction. Experimentswere carried out on Biacore T200 or Biacore 3000 SPR instruments.

As summarized in FIG. 17 , antibodies 20C1, 20B3, 8B11, 23H3, 18E9,14B6, OX40.1, and OX40.2 were ligand blockers; antibody 3F4 was apartial ligand blocker; and 14A2, 6E1, and OX40.5 were ligandnon-blockers.

Example 9: Biophysical Properties of OX40 Antibodies

The affinity of several OX40 antibodies for soluble human OX40 wastested by SPR analysis. Briefly, affinity measurements were carried outby capturing 1-10 μg/mL of the respective antibody on a CM5 chip coatedwith anti-human-CH1. Human-OX40-6×HIS antigen in either a singleconcentration of 400 nM or a 1:2 serial dilution from 400 nM was used.Experiments were carried out on BIACORE® T200 or BIACORE® 3000 SPRinstruments. Data was fit to a 1:1 model.

As shown in Table 13, the anti-OX40 antibodies tested had dissociationconstants (K_(D)s) in the range of 10⁻⁸ M to 10⁻⁹ M.

TABLE 13 K_(D) values for OX-40 antibodies Clone K_(D) (M) k-on (1/Ms)k-off (1/s) 3F4 7.13e−9 5.31e4 3.79e−4 8B11 1.05e−8  4.8e4  5.1e−4 14B68.84e−9  7.4E4 6.54e−4 6E1  1.1e−8 1.28e5 1.41e−3 14A2 1.51e−9 1.46e5 2.2e−4 18E9 2.04e−9 6.83E4 1.39e−4 20B3 3.71e−9 5.42e4 2.01e−4 23H3 3.6e−9  1.1E5 3.95e−4 20C1 3.22e−9 6.48E4 2.09e−4 OX40.21 1.49e−9 9.41e+5 0.0014

The thermal stability of the OX40.21 antibody was also tested, withresults summarized in Table 14. Thermal stabilities were determinedusing GE Healthcare CAP-DSC. Samples were run at 250 μg/mL concentrationin PBS. The scan rate was 60° C./hr. Data was fit to a non-2-statemodel. The OX40.21 antibody was determined to be one of the more stableantibodies tested when considered together with other attributes (e.g.,low off-target effects, immunogeneciity, etc).

TABLE 14 % Clone Tm1 Tm2 Tm3 reversibility 3F4 68 83 8B11 72.7 82.9 14B666.3 70.5 18E9 65.8 71.2 23H3 72.3 82.7 20C1 68.0 83.0 OX40.21 72.2 79.548% at 80° C.

The pharmacokinetics of the OX40.21 antibody after single intravenousdosing to cynomolgus monkeys also was tested. The OX40.21 antibodyexhibited acceptable pharmacokinetic (PK) properties after singleintravenous (IV) dosing to cynomolgus monkeys with linear PK (0.4 to 4mg/kg) and a long terminal half-life (6 days).

TABLE 15 Pharmacokinetic parameters of OX40.1 after intravenousadministration in cynomolgus monkeys (N = 3) Dose AUC(INF)* t_(1/2) CLTVss (mg/kg) μg/mL × day) (day) (mL/h/kg) (mL/kg) 0.4 86 ± 5  5.6 ± 0.50.20 ± 0.01 36 ± 2  4 785 ± 138 6.2 ± 0.6 0.22 ± 0.04 49 ± 12 PKparameters were calculated by a non-compartmental method. Values aremean ± SD. *PK parameters were calculated using plasma conc. up to 10days, except monkey 2 at 0.4 mg/kg up to 7 days *% AUCextra rangedbetween 24% and 42%

The human PK parameters of OX40.21 were projected from cynomolgus monkeyPK data using allometric scaling (assuming power exponent=0.85 for CLTand 1 for Vss). The projected human t_(1/2) was 10 days (Table 16). PKparameters were calculated by a two-compartment method.

TABLE 16 Projected Human Pharmacokinetic Parameters of OX40.21 DoseAUC(INF) t_(1/2) CLT Vss Ab (mg/kg) (μg/mL × day) (day) (mL/h/kg)(mL/kg) OX40.21 1 303 10 0.14 47

Example 10: FACS Cross-Blocking of PE-Labeled Anti-OX40 Antibody CloneL106 by a Panel of Unlabeled Anti-OX40 Antibodies

Several anti-OX40 antibodies were tested for their ability to block thebinding of PE-labeled anti-OX40 antibody clone L106 to humanOX40-transfected 293 cells. Briefly, 293 cells stably transfected withhuman OX40 were first incubated with varying concentrations of unlabeledanti-OX40 antibodies. The cells were then washed and incubated with afixed concentration of 2.5 μg/mL of PE-labeled L106 antibody (BDBiosciences). The fluorescence intensity of the staining was measuredusing a FACSCanto II flow cytometer (Becton Dickinson). The geometricmean fluorescence intensity (GMFI) of PE-L106 antibody staining for thecell population was calculated (FACSDiva software). Dose-response curvesfor blocking of L106 binding were generated using GraphPad Prismsoftware.

As shown in FIG. 18 , the 18E9 and OX40.1 antibodies fully blocked L106binding to human OX40-transfected cells, while 20B3 showed partialblocking. The remaining antibodies showed little or no blocking,indicating that 18E9 and OX40.1 bind to a different epitope on OX40 thanthe other antibodies tested.

Example 11: Cross-Block Analysis of Anti-OX40 Antibodies

This experiment was performed to test the cross-blocking properties ofvarious anti-OX40 antibodies to assess binding specificities. In brief,the OX40 antibody OX40.1 was conjugated to allophycocyanin (APC), andhuman OX40 antibodies OX40.4 and OX40.5 were biotinylated. A panel ofunconjugated human OX40 antibodies were applied in dose to engineered293 or HT1080 cell lines that over-express human OX40 protein on theirsurface and were permitted to bind at 4° C. for 30 min. Without washoutof the unconjugated Ab, APC-OX40.1 (1 μg/mL), biotin-OX40.4 (0.4 μg/mL),or biotin-OX40.5 (0.4 μg/mL) was applied to the assay wells and allowedto bind at 4° C. for 30 min. Cells were washed and if necessary furtherincubated in the presence of streptavidin-APC conjugate under the sameconditions. After the final wash, cells were analyzed on a FACSCantoflow cytometer (BD Bioscience, San Jose, Calif.). Mean fluorescenceintensity (MFI) signal was proportional to bound conjugated antibody.

As shown in FIGS. 19A-19C, binding of APC-OX40.1 to cells overexpressinghuman OX-40 protein was blocked by OX40.2 and OX40.5, but only modestly,if at all, by OX40.4. Binding of APC-OX40.1 was blocked by 8B11.H9,3F4.G11, 20B3.G2, and 14B6.C5, but not by 6E1.A12 and 14A2.B9. A diagramof the observed binding relationships between the antibodies evaluatedin FIGS. 19A-19C is shown in FIG. 19H.

FIGS. 19D-19E show that binding of biotin-OX40.4 was strongly blocked by20B3.G2, moderately blocked by 20C1.F2, weakly blocked, if at all, by3F4.G11 and 23H3.C6, and was not blocked by 14A2.B9. Binding ofbiotin-OX40.5 was strongly blocked by 20B3.G2, 23H3.C6, and 20C1.F2,moderately blocked by 3F4.G11, and weakly blocked by 14A2.B9.

FIGS. 19F-19G show that binding of biotin-OX40.4 was not blocked byOX40.1 or OX40.8, and was only weakly blocked, if at all, by OX40.5 orOX40.6. Binding of biotin-OX40.5 was blocked by OX40.1, moderatelyblocked by OX40.6, and was only very weakly blocked, if at all, byOX40.4 or OX40.6.

A diagram of the observed binding relationships between the Absevaluated in FIGS. 19D-19G is shown in FIG. 19I.

Example 12: Anti-OX40 Antibodies Bind to a Conformation Epitope/EpitopeMapping

This example shows that OX40.21 binds to non-denatured human OX40, butnot to denatured human OX40, and that binding is not affected byN-glycosylation.

Binding of OX40.21 to native or denatured OX40 that has N-linkedglycosylation or not was determined as follows. Samples of native (i.e.,non-denatured) and denatured human OX40 were incubated with or withoutthe enzyme N-glycanase PNGase F to remove N-glycosylation. Samples ofnative human OX40 with or without N-linked glycosylation were subjectedto SDS gel electrophoresis, and samples of denatured human OX40 with orwithout N-linked glycosylation were subjected to denaturing SDS gelelectrophoresis.

As shown in FIG. 20A, OX40.21 binds only to native OX40, and not to thedenatured form, and the presence or absence of glycosylation does notaffect binding to OX40. FIGS. 20B and 20C show that two N-glycopeptideswere identified by peptide mapping after deglycosylation (60% occupancyfor both AspN118 and AspN12).

These data suggest that OX40.21 binds to an epitope that isconformational and independent of N-linked glycosylation.

Epitope mapping studies were also conducted using mass spectrometry.Peptide fragments of his-tagged human OX40 (“hOX40”) were generated byenzymatic digestion with endoproteinases. LC-MS was performed using ABSciex 5600 Triple-TOF.

As shown in FIGS. 20D and 20E, binding experiments from native hOX40 bylimited proteolysis revealed that OX40.16 and OX40.21 boundpredominantly to the peptide DVVSSKPCKPCTWCNLR (SEQ ID NO: 178), whichcorresponds to amino acids 46-62 of the extracellular portion of maturehuman OX-40 (SEQ ID NO: 2). OX40.8 bound to the peptideDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179), which correspondsto amino acids 89-124 of the extracellular portion of mature human OX40(SEQ ID NO: 2). The location of the epitope bound by OX40.21 overlapspart of the binding site of OX40 ligand as determined by crystalstructure of the human OX40/OX40L complex (Protein Data Bank (PDB) IDcode 2HEV). Additional peptides identified by mass spectrometry forOX40.21 are shown in the upper panel of FIG. 20E, and includeQLCTATQDTVCR (SEQ ID NO: 184), SQNTVCRPCGPGFYN (SEQ ID NO: 185),SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182), and PCKPCTWCNLR (SEQID NO: 183).

Example 13: Anti-OX-40 Antibodies Promote T Cell Proliferation andInduce IFN-γ and IL-2 Secretion from T Cells

Anti-OX-40 antibodies were tested for their ability to induce T cellactivity in vitro by measuring the proliferation of and amount of IL-2and IFN-γsecreted by T cells incubated with the antibodies.

A transfected CHO cell line was generated for use as artificialantigen-presenting cells in a primary T cell activation assay. TheCHO-CD3-CD32A cell line expresses anti-human CD3 antibody in asingle-chain Fv format, along with the human Fc receptor CD32A topresent anti-OX40 antibodies on the CHO cell surface. Briefly, humanprimary CD4 T cells were isolated by negative selection (RosetteSep™,StemCell Technologies) and co-cultured with irradiated CHO-CD3-CD32Acells at an 8:1 T:CHO ratio, in the presence of graded doses ofanti-OX40 antibodies or isotype control antibody. After 3 to 4 days inculture at 37° C., supernatants were harvested for assessment of T cellactivation by means of measurement of secreted human IFNγ either byELISA (BD Biosciences) or HTRF assay (Cisbio), following themanufacturers' recommendations. Afterwards, tritiated thymidine wasadded for the final approximately 18 hours of culture to measure Tproliferation by tritiated thymidine incorporation, as an additionalassessment of T cell activation.

As shown in FIGS. 21A-21D and 22A-22D (and summarized in Table 17below), most tested anti-OX40 antibodies strongly potentiated human CD4T cell activation stimulated by CHO-CD3-CD32 cells, in a dose-dependentmanner, as measured by proliferation and IFNγ secretion. The panel ofantibodies tested in this assay co-stimulated T cell activation at leastas well as, or better than, OX40.1, OX40.4, and OX40.5.

TABLE 17 Proliferation EC₅₀ (nM) IFNγ EC₅₀ (nM) Name (mean ± SD) n (mean± SD) n 3F4 0.016 ± 0.008 5 8B11 0.022 ± 0.027 3 18E9 0.010 ± 0.005 320B3 0.008 ± 0.003 3 20C1 0.008 ± 0.006 4 23H3 0.028 ± 0.017 3 6E1 0.014± 0.008 2 14A2 0.037 ± 0.044 4 14B6 0.012 ± 0.008 3 OX40.6 0.032 ± 0.0282 0.033 ± 0.004 2 OX40.8 0.043 ± 0.037 2 0.024 1 OX40.16 0.017 1 0.044 1OX40.17  0.009± 1 0.044 1 OX40.18  0.230± 1 0.490 1 OX40.21 0.011 ±0.006 9 0.043 ± 0.023 9 OX40.1 0.024 ± 0.012 4 OX40.4 0.094 1 ~2.3e+0091 OX40.5 1.900 1 ~37 1

The anti-human OX40 antibodies were also tested for their effects onstimulating primary T cells in cultures of staphyloccus enterotoxin B(SEB)-activated human peripheral blood mononuclear cells (PBMCs). Humanwhole blood was obtained from AllCells, Inc. (Berkeley, Calif.) or fromdonors at Bristol-Myers Squibb, Redwood City, Calif. under the auspicesof an in-house phlebotomy program. PBMCs were isolated by gradientpurification on a Ficoll-Hypaque cushion and cultured for 3 days inculture medium supplemented with fixed, suboptimal (85 ng/mL) ofsuperantigen staphylococcus enterotoxin B (SEB; Toxin Technologies,Sarasota, Fla.) in the presence of graded doses of OX40 antibodies orisotype control antibody together with 2-5 μg/mL of solublecross-linking antibody, F(ab′)2 goat anti-human Fcγ. After culturing for3 days at 37° C., supernatants were harvested for assessment of T cellactivation by means of ELISA measurement of secreted human IL-2.Briefly, culture supernatants were diluted 1:10 in sample diluent andtested for the presence of human IL-2 by ELISA (BD Bioscience) per themanufacturer's recommended protocol. Following the addition of TMBsubstrate, assay plates were read on a Spectramax 340PC reader usingSoftmax operating software at a wavelength of 650 nm. Measured opticaldensities of the chromogenic substrate were proportional to bounddetecting antibody.

Data from PBMCs isolated from different donors are shown in FIGS.23A-23F. In general, solubly-crosslinked clone 20C1.F2 elicited a morerobust cytokine response (EC50 of 1.3-2.0 nM) compared tosolubly-crosslinked clones 23H3.C6, 8B11.H9, 3F4.G11, 18E9.G5, 6E1.A12,and 20B3.G2 (FIGS. 23A-23C). With respect to antibodies with variableregion mutations, OX-40.21, in general, elicited a more robust cytokineresponse compared to solubly-crosslinked antibodies OX-40.17, OX40.18,OX40.6, and OX40.8 (FIGS. 23D-23F). Data from these donors together withdata from 8 additional donors, in which additional anti-OX40 antibodieswere tested, collectively demonstrate that on average, OX40.21 exhibitedsuperior potency in enhancing T cell responses compared to OX40.1,OX40.2, OX40.4, OX40.5, OX40.17, and OX40.18. These results furtherdemonstrate that OX40.21 elicits responses that are comparable to thoseelicited by OX40.6 and OX40.8 (Table 18).

TABLE 18 Donor # OX40.21 OX40.17 OX40.18 OX40.6 OX40.8 BMS-009 EC50 (nM)0.34 0.97 3.21 0.45 0.50 BMS-012 EC50 (nM) 0.29 1.74 ~1.67 0.66 0.09BMS-016 EC50 (nM) 0.04 1.40 >100 0.29 0.09 Donor # OX40.21 OX40.17OX40.18 OX40.6 OX40.8 OX40.1 OX40.2 OX40.4 OX40.5 WB10024 EC50 (nM) 0.821.06 ~3.17 ~0.42 ~0.44 1.87 2.53 1.25 ~1.83 WB10025 EC50 (nM) 1.31 1.35~1.14 ~0.85 0.79 2.08 ~3.26 1.08 3.07 WB10026 EC50 (nM) 0.79 1.68 3.020.28 0.63 2.24 3.16 1.14 2.59 WB10027 EC50 (nM) 1.08 1.15 ~3.27 0.45~0.46 1.26 2.26 0.93 ~3.09 Donor # OX40.21 OX40.17 OX40.18 OX40.6 OX40.8OX40.1 OX40.2 OX40.4 OX40.5 WB10137 EC50 (nM) 0.56 0.92 >100 0.56 0.42~1.824 >100 1.37 >100 BMS-001 EC50 (nM) 0.41 0.41 ~84.76 0.49 ~0.42 0.644.95 ~0.89 ~0.43 BMS-004 EC50 (nM) ~0.84 1.03 2.43 0.48 0.55 2.52 13.890.84 1.94 BMS-015 EC50 (nM) 0.76 1.16 >100 ~0.46 ~0.42 2.41 >100 0.911.92 Mean EC50 (nM) 0.64 1.17 2.89 0.46 0.44 1.86 5.36 1.07 2.38 *Eachset of experiments was performed on different days.

Example 14: Anti-OX40 Antibody Promotion of NK92-Mediated Cell LysisUsing Cell Lines

Several anti-human OX40 antibodies were tested for their ability topromote NK92 cell-mediated lysis of activated CD4⁺ T cells using calceinrelease as a read-out. Briefly, CD4⁺ T cells for use as target cellswere separated by negative selection using magnetic beads and activatedfor 72 hours with beads coated with anti-CD3 and anti-CD28. After threedays, NK92 cells were plated with calcein AM-labeled activated CD4⁺cells at a ratio of 5 to 1. A titration of each anti-OX40 antibody wasadded and cells were incubated for two hours. Calcein release wasmeasured by reading the fluorescence intensity of the media using anEnvision plate reader (Perkin Elmer). The percentage ofantibody-dependent cell lysis was calculated based on mean fluorescenceintensity (MFI) with the following formula: [(test MFI−meanbackground)/(mean maximum−mean background)]×100.

As shown in FIG. 24 , OX40.8 and OX40.16 induced the highest amount ofspecific lysis of target cells (60% and 30%, respectively). The EC₅₀ ofOX40.8 was 16 ng/mL and that for OX40.16 was 4 ng/mL. All otherantibodies tested induced ADCC at levels too low for accuratequantitation.

Example 15: Anti-OX40 Antibody Promotion of NK-Mediated Cell Lysis ofPrimary Human CD4+ T Cells

Several anti-human OX40 antibodies were tested for their ability topromote primary NK cell-mediated lysis of activated CD4⁺ T cells.Briefly, CD4⁺ T cells for use as target cells were separated from PBMCsfrom two donors by magnetic selection and activated for 72 hours withbeads coated with anti-CD3 and anti-CD28. NK cells, for use aseffectors, were separated from a separate donor by negative selectionusing magnetic beads and activated with IL-2 for 24 hrs. Following theactivation period, NK effector cells were mixed with calcein-labeledtarget T cells at 20:1, 10: 1, or 5:1 ratios in the presence of antibodyat 1 μg/ml for 2 hours. The level of calcein released by lysed targetcells was measured by reading the fluorescence intensity of the mediausing an Envision plate reader (Perkin Elmer). The percentage ofantibody-dependent cell lysis was calculated based on mean fluorescenceintensity (MFI) with the following formula: [(test MFI−meanbackground)/(mean maximum−mean background)]×100.

As shown in FIGS. 25A and 25B, activated CD4+ T cell targets from twodonors were lysed most effectively by OX40.8. Lower levels of ADCCactivity were seen with both OX40.21 and OX40.1.

Example 16: OX40 Antibody Promotion of Macrophage-Mediated CellPhagocytosis of OX40-Expressing HEK293 Cells

To determine the antibody-mediated phagocytic activity of several OX40antibodies, primary human macrophages were cultured for four hours withCellTrace Violet-labeled HEK293/OX40 cells and a titration of anti-OX40antibodies. After four hours, cells were harvested, stained withanti-CD64-APC, and run on a flow cytometer. Cells that stained doublepositive for CD64 and CellTrace Violet were considered to have beenphagocytosed. The percentage of target cells phagocytosed was calculatedusing the formula: 100×(Number of double positive cells/Total number ofCellTrace Violet positive cells).

As shown in FIG. 26 , all tested anti-OX40 antibodies induced thephagocytosis of OX40-expressing target cells in a dose-dependent manner.OX40.8 had the highest overall level of phagocytosis and the lowest EC₅₀concentration of 6.2 ng/mL. This demonstrates that human IgG1 anti-OX40antibodies induce FcR-mediated phagocytosis in a dose-dependent manner.

Example 17: Anti-OX40 Antibodies Bind the C1q Component of HumanComplement

A colorimetric ELISA assay was developed to evaluate whether the C1qcomponent of human serum complement binds to the OX40.21 antibody. Alltested antibodies were coated on a high binding immunoassay plate at 10μg/mL. After blocking unoccupied protein binding sites, graded doses ofhuman C1q (3.125-200 μM) were added to the wells, including blockedempty wells that served as controls for non-specific background C1qbinding to the assay plate. Binding of C1q to the immobilized antibodieswas detected using a combination of biotinylated mouse anti-C1q antibodyand streptavidin-poly-HRP, together with tetramethylbenzidine substrate.The results are reported as the optical density read at 450 nm minus 630nm.

As shown in FIG. 27 , C1q bound to OX40.21 (solid squares) and the humanIgG1 isotype control (open circles) in a dose dependent manner. Thelevel of C1q binding to OX40.21 however, was lower than to the humanIgG1 isotype control antibody. As expected, there was little backgroundsignal (gray circles) and no evident C1q binding to an IgG1.1 isotypecontrol (solid black circles). The IgG1.1 antibody contains fivemutations in the Fc portion designed to eliminate C1q binding and FcRinteraction. This result demonstrates that the C1q component of humanserum complement can bind to OX40.21 and indicates that OX40.21 mayinduce complement mediated lysis of OX40-expressing cells in vivo.

Example 18: OX40 is Expressed in Tumor Infiltrating Lymphocytes

OX-40 is expressed in tumor infiltrating lymphocytes, with a patternthat is generally limited to CD4+ cells (FIG. 28A) with minimalexpression on CD8+ T cells (FIG. 28B) in colorectal, lung, and ovariancancer (FIG. 28C).

Similarly, OX40 is expressed by CD4+ T cells and Tregs in mouse Sa1Ntumors (FIG. 28D) and mouse MC38 tumors (FIG. 28E). To test expressionof mouse OX-40 in tumors, 2×10⁶ SA1N sarcoma cells or 2×10⁶ MC38 cellswere implanted subcutaneously into AJ or B6 mice respectively. On day 15post-implantation, tumors were harvested, dissociated into single cellsuspensions, and stained for flow cytometry. T cell populations wereidentified based on their expression of CD8, CD4 and Foxp3. For Sa1Ntumors, CD4+ Foxp3+ cells from the tumor are shown in the red histogram,CD4+ Foxp4− cells are in the blue histogram and CD8+ cells are in theorange histogram (FIG. 28D). Isotype control stained cells are in thegreen histogram. For MC38 tumors, Tregs are shown in the blue histogram,CD4+ cells are shown in the green histogram and CD8+ cells in the redhistogram (FIG. 28E).

Example 19: Anti-OX40 Antibody Reversal of Treg Cell-MediatedSuppression

Several anti-human OX40 antibodies were tested for their ability toreverse regulatory T (Treg) cell-mediated suppression of human CD4⁺ Tcell proliferation. Briefly, Treg and T responder (Tresp) cells wereisolated by enriching PBMCs for CD4⁺ cells by magnetic bead separationand then sorting CD4⁺CD25^(hi)CD127^(lo) Treg andCD4⁺CD25^(lo)CD127^(hi)CD45RO⁺ Tresp cells. Tresp cells were thenlabeled with proliferation dye and plated with titrating numbers of Tregcells, beginning at a 1:1 ratio. Cultures were stimulated with 3 μg/mLplate-bound anti-CD3, 1 μg/mL soluble anti-CD28, and 2 μg/mL plate-boundanti-OX40 or isotype control. After 96 hours, Tresp cell proliferationwas measured by assessing dye dilution using flow cytometry.

As shown in FIG. 29 , in both the presence and absence of Treg cells,the anti-OX40 antibodies increased Tresp cell proliferation compared tothe isotype control. This suggests that the anti-OX40 antibodies testedreversed the suppressive effects of Treg cells on Tresp cellproliferation.

Example 20: Toxicity Studies

OX40.6 (2 mg/kg) was administered intravenously to monkeys on Days 1(FIG. 30A) and 29 (FIG. 30B) to evaluate any associated toxicities. Noevidence of tolerability issues or clinical pathology abnormalities wasobserved. OX40.6 stimulated an enhanced immune response to KLH, ascharacterized by a trend towards enhanced CD69 expression in CD4+ Tcells in an ex vivo KLH recall assay. Two of 4 monkeys exhibitedaccelerated clearance, which correlated with the formation of anti-drugantibodies.

The concentration of OX40.6 in cynomolgus monkey serum samples for theexperiment above was analyzed by a chemiluminescence (CL) immunoassay.OX40. 6 antibody was used to prepare calibrators and quality control(QC) samples. Biotinylated-human-OX40-his was immobilized onstreptavidin-coated microplates (Greiner Bio-one) as a capture moleculefor OX40.6. Samples, standards, and quality control samples brought upto a final matrix of 10% cyno serum were incubated on the plates.Samples were analyzed at 10% minimum required dilution in 1%BSA/PBS/0.05% Tween 20 (PTB) containing 2% mouse serum. The unboundmaterial was washed away and the captured OX40.6 antibody was detectedusing an HRP-labeled mouse monoclonal anti-human IgG antibody as thedetection molecule. Following addition of SuperSignal ELISA PicoChemiluminescent Substrate (Thermo Scientific), the concentration ofOX40.6 in cyno serum samples was calculated from luminescence intensityas measured by a M5 plate reader using a 4-parameter logistic (4-PL)calibration curve generated from OX40.6 antibody calibrators. The rangeof the OX40.6 antibody calibration curve was from 5 to 5,000 ng/mL incyno serum. The upper and lower limits of quantification were 5,000 and10 ng/mL, respectively (i.e., ULOQ 5000 ng/mL, LLOQ 10 ng/mL). Qualitycontrol samples were prepared at 3750, 400, and 20 ng/mL in cynomolgusmonkey serum and analyzed on each plate to ensure acceptable assayperformance. Calibrators, QCs, and samples were diluted 5-fold in PTBcontaining 2% mouse serum. Four streptavidin plates were used to analyzethe samples. Assay performance was within an acceptable range:interplate % CV of standards was below 25%, and QC recovery was within±30% nominal values.

The presence of anti-drug antibodies to OX40.6 in cynomolgus monkeyserum in the experiment described above was determined byelectrochemiluminescence (ECL) bridging immunoassay. Specifically, mousemonoclonal anti-human IgG Fc antibody was used to prepare the positivecontrol. Biotinylated-anti-OX40: anti-hOX40-his was used at 25 ng/mL asthe capture molecule and ruthenylated-anti-OX40: anti-hOX40-his was usedat 25 ng/mL as the detection molecule. Samples were analyzed at 100-folddilution in 1% BSA/PBS/0.05% Tween 20 (PTB) containing capture anddetection molecules. After 2 hours of incubation in polypropyleneplates, the sample mix was transferred to streptavidin-coated MSDplates. Following a one hour incubation, unbound material was washedaway, MSD read buffer was added, and ECL was measured with the MSD platereader SI6000. The positive control (Mouse anti-human IgG Fc) wasprepared at 1000 (HPC), 100 (MPC), and 10 ng/mL (LPC) in cynomolgusserum. Pooled cynomolgus serum was used as a negative control (NC). Thesignal ratio for HPC, MPC, and LPC versus NC was 102, 10, and 2,respectively. One streptavidin plate was used to analyze the samples.Assay performance was within the acceptable range: % CV of the PC wasbelow 10%, and the raw signal for the negative control (54 RLU) wascomparable to the raw signal for predose samples (48-55 RLU).

Example 21: Immunogenicity Risk Assessment Study

In vitro T cell proliferation assays were conducted for several of theanti-human OX40 antibodies to assess their human immunogenicitypotential. Briefly, peripheral blood mononuclear cells (PBMC) fromhealthy volunteers were isolated by Ficoll (GE Healthcare) and gradientcentrifugation, and human lymphocyte antigen (HLA) Class II wascharacterized by polymerase chain reaction (PCR) amplification andhybridization with oligonucleotide probes (ProImmune).

A panel of 40 PBMC donors having HLA Class II types closely matchingworld population frequencies was used for an assay run. PBMCs werelabeled with CFSE (Invitrogen) to monitor proliferation and plated on 96well plates in 6 replicates at 200,000 cells per well in RPMI (Lonzo)containing 10% human AB (Bioreclamation), non essential amino acids(Gibco), and pen-strep (Gibco). Anti-human OX40 antibodies, controlsproteins, reference antibodies, and ConA were cultured with PBMCs at 1μM for 7 days, after which media was washed away and cells were labeledwith an anti-human CD4 APC (BD science) monoclonal antibody. Afterremoval of unbound anti-CD4 antibody with a wash step, cells were fixedwith 3.7% formalin (Sigma) in PBS, and analyzed by flow cytometry todetermine the percentage of proliferating CD4+ cells.

The percentage of 40 donors that showed a positive response (defined asa significant increase in proliferating CD4+ T cells relative tomedia-incubated PBMCs) for the different anti-human OX40 antibodies isshown in FIG. 31 . All variants of the anti-human OX40 antibodies showedlow potential to activate CD4+ cells in this assay, comparable to thelow QC protein, with the exception of OX40.16 and OX40.21, which did notshow a positive CD4 proliferation response in any of the 40 donors.These results suggest that these anti-human OX40 antibodies have lowpotential to elicit an anti-drug antibody response in humans.

Example 22: Binding to Activated Fc Receptors Enhances Anti-mOX40Activity in a Colon Carcinoma Model

To test the role of FcR binding in the activity of anti-mouse OX40antibodies in mouse tumor models, anti-OX40 antibodies of differentisotypes were tested. C57BL/6 mice were subcutaneously injected with 2million MC38 tumor cells. After 7 days, tumor volumes were determinedand mice were randomized into treatment groups so as to have comparablemean tumor volumes. Antibodies formulated in PBS were administeredintraperitoneally on days 7, 10, and 14 at 200 μg per dose in a volumeof 200 μl.

In syngeneic mouse tumor models, anti-murine OX40 antibodies (e.g.,OX86, rat IgG1) exhibit anti-tumor activity. Since varying the isotypeof many antibodies specific for T cell surface receptors (bothco-stimulatory and co-inhibitory) can alter the anti-tumor activity ofthese antibodies, mouse Fc isotype variants of OX86, an antibody whichdoes not block the OX40/OX40L interaction, were generated. As shown inFIGS. 32A-32C, OX86 formatted as a mouse IgG2a Fc (FIG. 32C) results insuperior anti-tumor activity compared to OX86 formatted as a mouse IgG1(FIG. 32B). This is likely due both to depletion of Treg cells at thetumor site and to T effector cell expansion from antibody-mediatedagonism of OX40.

To confirm the effects of different antibody isotypes on tumorinfiltrating T cell populations, tumors from MC38 mice that were treatedwith the different isotypes were assessed by flow cytometry. Selectedmice were sacrificed and tumors and spleens were harvested for analysison day 15 after tumor implantation. Single cell suspensions wereprepared by dissociating tumor and lymph node with the back of a syringein a 24 well plate. Cell suspensions were passed through 70 μm filters,pelleted, resuspended, and counted. Cells were then plated in 96 wellplates with 1×10⁶ cells per well for staining. Samples were thenanalyzed on a FACS Canto flow cytometer (BD). Analysis of the spleensand tumors of tumor bearing mice treated with anti-OX40 antibodies showthat the IgG2a isotype can deplete CD4+ Tregs in tumors (FIG. 33A), andthat IgG1 and IgG2a isotypes can activate T cell expansion in theperiphery (FIG. 33B) and result in increased cell numbers in the spleen(FIG. 33C). These results suggest that agonism of OX40 (but notnecessarily blocking the OX40/OX40L interaction) and Fc receptor bindingof the OX86 antibody promotes anti-tumor activity.

The role of human Fc and FcRs were tested using mice where mouse FcRshave been knocked out and replaced with the human FcRs. Theseexperiments were performed using a bone marrow chimera system whereCD45.1 congenic hosts were irradiated then reconstituted with human FcRtransgenic bone marrow cells. These mice were then allowed toreconstitute for 8 weeks before being inoculated with 2×10⁶ MC38 tumorcells. After 7 days, tumor volumes were determined and mice wererandomized into treatment groups so as to have comparable mean tumorvolumes. Antibodies formulated in PBS were administeredintraperitoneally on days 7, 10, and 14 at 200 μg per dose in a volumeof 200 μl. Mice were treated with either a control human IgG1 (FIG.34A), a chimeric OX-86 human G1 hybrid Ab (FIG. 34B), or the OX-86 humanG1 hybrid with a S267E mutation (FIG. 34C). The results were similar towhat was observed with the mouse isotypes, i.e., the human IgG1 antibodyhad a significant anti-tumor effect as it can bind to activating FcRs,while the S267E mutation which increases binding to both CD32B and CD32Ahad higher activity (FIGS. 34A-34C). This higher level of activity islikely due to increased agonism on effector T cells as well as increaseddepletion of Tregs at the tumor site.

T cell populations at the tumor site and spleens of tumor bearing micewere examined as described earlier. Tregs were less prevalent in micetreated with either the G1 or G1 S267E antibody, with a larger effectseen with the S267E isotype (FIG. 35A). Increases in the percentages ofCD8+ T cells (FIG. 35B) and CD4+ effector (FIG. 35C) and at the tumorsite were also evident, and these increases were greater with the G1S267E antibody. Increased cellularity in the spleens of mice treatedwith the anti-OX-40 antibodies was also noted (FIG. 35D). These resultssuggest that the OX86-hIgG1 antibody exhibited potent anti-tumoractivity (FIGS. 34A-34C) and measurable Treg depletion (FIGS. 35A-35D).

Example 23: A Blocking Anti-OX40 Antibody Exhibits Anti-Tumor Activityin a Mouse Tumor Model

The following experiment was conducted to determine whether an antibodywhich blocks the interaction between OX40/OX40L exhibits potentanti-tumor activity. To this end, a hamster anti-mouse OX40 antibodywhich blocks the OX40/OX40L interaction (hamster IgG1 8E5 antibody) wasgenerated and tested for its anti-tumor activity in a subcutaneous mouseCT-26 tumor model. CT-26 is a mouse colon adenocarcinoma tumor cell linewhose solid tumor growth can be monitored in BALB/c mice when the cellsare transplanted subcutaneously.

Female BALB/c mice (Charles River Laboratories, Hollister, Calif.) wereacclimated for a minimum of three days prior to the start of thestudies. Mice were housed 5 animals per cage, and the cages were placedin microisolator ventilated racks. Housing was at 18-26° C. and 50+20%relative humidity with at least twelve room air changes per hour. A 12 hlight/dark cycle was maintained. Animals were provided with sanitizedlaboratory rodent diet and municipal water ad libitum.

CT-26 cells were maintained in RPMI-1640 medium (Hyclone, Cat. No.SH30096.01) supplemented with 10% fetal bovine serum (FBS; Hyclone, Cat.No. SH30071.03). Approximately twice a week, cells contained in a singleT175 flask were divided and expanded to four T175 flasks at a 1:5dilution until sufficient number of cells were obtained for tumorimplantation. The cells were harvested near 80% confluence, washed andresuspended in PBS.

On Day 0, 1×10⁶ CT-26 cells were implanted into the mice using a 1 ccsyringe (Becton Dickinson, Franklin Lakes, N.J.) and 27 gauge ⅝ inchneedle. Tumors were then measured two times weekly in 3 dimensions withan electronic caliper (Mitutoyo, Aurora, Ill.) and recorded. Tumorvolumes (mm³) were calculated using the formula:width×length×height×0.5. Following tumor volume measurements on Day 6post implantation, mice were staged according to tumor volume. Mice witha mean tumor volume of 26 mm³ were randomized into groups and treated asshown in Table 19.

The hamster isotype control antibody is an inert Armenian hamster IgGmonoclonal antibody (mAb) to GST (clone PIP, catalog #BE0260; BioXcell,West Lebanon, N.H.). It was prepared in PBS immediately prior toadministration to provide doses of 10 mg/kg per mouse viaintraperitoneal (IP) injection on Days 6, 10 and 14 as shown in Table19.

The monoclonal antibody against mouse OX40 (clone 8E5) was prepared inPBS immediately prior to administration to provide doses of 10, 3, 1 or0.3 mg/kg per mouse via IP injection on Days 6, 10 and 13 as shown inTable 19.

TABLE 19 Treatment Treatment Dose N Route schedule Hamster IgG 10 mg/kg12 IP Days 6, 10, 14 Istoype Control mAb Hamster anti-mouse OX40 10mg/kg 12 IP Days 6, 10, 14 (clone 8E5) mAb Hamster anti-mouse OX40 3mg/kg 12 IP Days 6, 10, 14 (clone 8E5) mAb Hamster anti-mouse OX40 1mg/kg 12 IP Days 6, 10, 14 (clone 8E5) mAb Hamster anti-mouse OX40 0.3mg/kg 12 IP Days 6, 10, 14 (clone 8E5) mAb

Animals were checked daily for postural, grooming, and respiratorychanges, as well as lethargy. Animals were weighed two times weekly andeuthanized if weight loss was ≥20%. Mice were checked for the presenceand size of tumors twice weekly until death or euthanasia. Tumors weremeasured in 3 dimensions with an electronic caliper (Mitutoyo, Aurora,Ill.) and recorded. Response to treatment compounds was measured as afunction of tumor growth. If the tumor reached a volume of ≥1500 mm³ orappeared ulcerated, animals were euthanized.

As shown in FIGS. 36A-36E, the hamster anti-mouse OX40 mAb (clone 8E5;FIGS. 36B-36E showing treatment with different doses of 8E5)demonstrated potent anti-tumor activity in the subcutaneous CT-26 modelas compared to the hamster IgG isotype control group (FIG. 36A). The 8E5antibody was administered at doses ranging from 0.3 to 10 mg/kg, andeven at the lowest dose evaluated (0.3 mg/kg), 10 of 12 mice weretumor-free (TF) at the end of the study period (Day 72). Although thenumber of tumor-free mice did not differ significantly amongst each dosegroup, with each group having 9 or 10 tumor-free mice by the end of thestudy period, mice treated with either of the two highest doses (3 or 10mg/kg) showed more tumor growth delays as compared to mice treated withthe two lowest doses (0.3 or 1 mg/kg). There were no tumor-free mice inthe isotype control-treated group; all mice in that group had beensacrificed by Day 39 as a result of ulceration or tumor burden (>1500mm³).

These data indicate that an anti-OX40 antibody that blocks theinteraction between OX40 and OX40-L demonstrates potent anti-tumoractivity in a subcutaneous mouse CT-26 tumor model when administered tomice with established tumors.

Example 24: OX40 Agonism Synergizes with PD-1 Blockage in a Murine MC38Colon Carcinoma Model

To test for synergy between anti-OX-40 antibody and anti-PD-1 antibodytreatments, combinations of these antibodies were tested in the MC38mouse tumor model. C57BL/6 mice were subcutaneously injected with 2million MC38 tumor cells. After 7 days, tumor volumes were determinedand mice were randomized into treatment groups so as to have comparablemean tumor volumes. Antibodies formulated in PBS were administeredintraperitoneally on days 7, 10, and 14 at 200 μg per dose in a volumeof 200 μl.

As shown in FIGS. 37A-37D, both the anti-PD-1 antibody (FIG. 37B) andanti-OX40 antibody (FIG. 37C) showed minimal activity when used alone,but had significant anti-tumor activity when combined (FIG. 37D), with 5of 8 mice rendered tumor free.

Example 25: OX40 Agonism Enhances the Response to Vaccines in CynomolgusMonkey

Enhancement of immune responses to vaccines was measured to evaluate theability of the OX40.6 antibody to stimulate immune responses incynomolgus monkeys. This approach was selected because the desiredeffect, i.e., enhancement of immune responses to tumors, cannot beevaluated in healthy non-human primates, as they lack tumors.

Monkeys were immunized with keyhole limpet hemocyanin (KLH) on Day 1 (10mg, intramuscularly) and with hepatitis B virus surface antigen (HBsAg)(ENGERIX-B) (20 μg intramuscularly on Days 1 and 29). Followingadministration of the vaccines, the monkeys were dosed intravenouslywith 0 or 2 mg/kg of OX40.6 antibody on Days 1 and 29. Immune responseswere measured on Days 22 and 41 by ex vivo T cell response to KLH and byT-cell-dependent antibody responses to KLH and HBsAg. As shown in FIGS.38A and 38B, OX40.6-related findings at 2 mg/kg at Days 22 (FIG. 38A)and 41 (FIG. 38B) included an increase in the ex vivo recall response toKLH, characterized by increases in the mean percent of CD69+,IFN-gamma+, and TNF-alpha+ expressing CD4+CD8− T cells.

Example 26: Fc Receptor Binding for Antibodies with Engineered ConstantDomains

This Example demonstrates that antibodies having modified heavy chainconstant regions comprising the CH1 and hinge of IgG2 bind to FcγRs whenthey contain CH2 and CH3 domains of IgG1.

In addition to antigen binding by the variable domains, antibodies canengage Fc-gamma receptors (FcgRs) through interaction with the constantdomains. These interactions mediate effector functions such asantibody-dependent cellular cytotoxicity (ADCC) and antibody-dependentcellular phagocytosis (ADCP). Effector function activity is high for theIgG1 isotype, but very low or absent for IgG2 and IgG4 due to theseisotypes having lower affinity for FcgRs. In addition, the effectorfunction of IgG1 can be modified through mutation of amino acid residueswithin the constant regions to alter FcgR affinity and selectivity.

The binding of antibodies to Fc gamma receptors (FcγRs or FcgRs) wasstudied using biosensor technologies including Biacore surface plasmonresonance (SPR) and Fortebio Biolayer Interferometry (BLI). SPR studieswere performed on a Biacore T100 instrument (GE Healthcare) at 25° C.The Fab fragment from a murine anti-6×His antibody was immobilized on aCM5 sensor chip using EDC/NHS to a density of ˜3000 RU. Varioushis-tagged FcgRs (7 ug/ml) were captured via the C-terminal his-tagusing a contact time of 30 s at 10 ul/min, and the binding of 1.0 uMantibody was evaluated in a running buffer of 10 mM NaPO4, 130 mM NaCl,0.05% p20 (PBS-T) pH 7.1. FcgRs used for these experiments included CD64(FcgRI), CD32a-H131 (FcgRIIa-H131), CD32a-R131 (FcgRIIa-R131), CD32b(FcgRIIb), CD16a-V158 (FcgRIIIa-V158), CD16b-NA1 (FcgRIIIb-NA1), andCD16B-NA2 (FcgRIIIb-NA2). BLI experiments were performed on a FortebioOctet RED instrument (Pall, Fortebio) at 250C in 10 mM NaPO4, 130 mMNaCl, 0.05% p20 (PBS-T) pH 7.1. Antibodies were captured out ofundiluted expression supernatants on protein A coated sensors, followedby the binding of 1 μM hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, or0.1 μM hCD64 analytes.

First, antibodies were made that contain modified IgG1 Fc domainsincluding the substitutions S267E (SE) and S267E/L328F (SELF), as wellas various combinations of the mutations P238D, P271G, H268D, A330R,G237D, E233D, referred to as V4, V7, V8, V9 and V12. The binding ofthese antibodies was studied by Biacore SPR with comparison to IgG1f,IgG2.3 (IgG2-C219S) and IgG4.1 (IgG4-S228P) antibodies, as well as anIgG1.1f antibody which has been engineered to reduce binding to allFcgRs. The results, which are shown in FIG. 39 , demonstrate theexpected FcgR binding properties for IgG1f, IgG2.3 and IgG4.1 and themutated IgG1 antibodies, including increased CD32a-H131, CD32a-R131 andCD32b binding for SE and SELF, as well as increased selectivity of theV4, V7, V8, V9 and V12 mutants for CD32b over CD32a-H131 and CD32a-R131(FIG. 39 ).

The next set of constructs was used to engineer effector function intothe otherwise effector function negative IgG2 isotype. For this study,the mutations described above were introduced in the context of IgG2.3constant region, or an IgG2.3/IgG1f hybrid termed IgG2.3G1-AY (Table20). Antibodies were expressed at small scale as supernatants, andtested for binding to FcgRs using Fortebio Octet BioLayer Interferometrybiosensor technology. Since the antibodies were present at lowconcentration in the supernatants, the experiment was performed bycapturing antibodies out of the supernatants using protein A coatedsensors, followed by binding of FcgR analytes in solution. Purified andsupernatant control IgG1f including wild type IgG1, SE, P238D, V4 andV12 antibodies were also included for comparison, and each of thesecontrol antibodies demonstrated expected FcgR binding properties (FIG.40 ). The IgG2.3 antibody also demonstrated the expected bindingprofile, with appreciable binding to only CD32a-H131. However, allmutations to introduce S267E, L328F, P238D, P271G, H268D, A330R, G237D,or E233D mutations into IgG2.3 failed to recapitulate the FcgR affinityof the corresponding engineered IgG1 mAbs (FIG. 40 ). In contrast, theIgG2.3G1-AY construct was able to fully preserve the FcgR bindingproperties of wild type IgG1, while retaining the CH1 and hinge regionsof IgG2.3. In addition, all IgG2.3G1-AY mutants containing S267E, L328F,P238D, P271G, H268D, A330R, G237D, and E233D demonstrated FcgR bindingproperties comparable to the IgG1 version mAbs containing the samemutations (FIG. 40 ). This demonstrates the successful engineering ofantibodies with CH1 and hinge regions of IgG2 combined with effectorfunction of wild type or mutant IgG1.

TABLE 20 Engineered IgG2 constructs Seq Set ID Construct ID# 1 IgG2.3hHC-IgG2-C219S 258 IgG2.3- hHC-IgG2- 267 V13 C219S - P238D IgG2.3-hHC-IgG2- 268 V14 C219S - P238D, P271G IgG2.3- hHC-IgG2- 269 V15 C219S -P238D, H268D, P271G IgG2.3- hHC-IgG2- 270 V16 C219S - P238D, P271G,A330R IgG2.3- hHC-IgG2- 271 V17 C219S - P238D, H268D, P271G, A330RIgG2.3- hHC-IgG2- 272 V18 C219S - S267E IgG2.3- hHC-IgG2- 273 V19C219S - S267E, L328F 2 IgG2.3G1 hHC4gG2- 262 C219S/hHC4gG1f IgG2.3G1-hHC4gG2- 274 AY-V20 C219S/hHC4gG1f - P238D IgG2.3G1- hHC4gG2- 275 AY-V21C219S/hHC4gG1f - P238D, P271G IgG2.3G1- hHC4gG2- 276 AY-V22C219S/hHC4gG1f - P238D, H268D, P271G IgG2.3G1- hHC4gG2- 277 AY-V23C219S/hHC4gG1f - P238D, P271G, A330R IgG2.3G1- hHC4gG2- 278 AY-V24C219S/hHC4gG1f - P238D, H268D, P271G, A330R IgG2.3G1- hHC4gG2- 279AY-V25 C219S/hHC4gG1f - G237D, P238D, H268D, P271G, A330R IgG2.3G1-hHC4gG2- 280 AY-V26 C219S/hHC4gG1f - E233D, G237D, P238D, H268D, P271G,A330R IgG2.3G1- hHC4gG2-C219S/ 266 AY-V27 hHC4gG1f - S267E IgG2.3G1-hHC4gG2-C219S/ 281 AY-V28 hHC4gG1f - S267E, L328F

This engineering strategy was further explored by producing otherantibodies formatted with IgG2.3G1-AY, IgG2.3G1-AY-S267E(IgG2.3G1-AY-V27), as well as IgG2-B-form variants (IgG2.5G1-AY andIgG2.5G1-AY-V27), and other hybrid antibodies containing differentcombinations of IgG1 and IgG2 constant domains, and testing the bindingof these antibodies to anti-his Fab captured his-tagged FcgRs usingBiacore SPR technology. In agreement with the Octet supernatant data,the SPR data showed that the IgG2.3G1-AY and IgG2.3G1-AY-V27 antibodieshad comparable FcgR binding properties to IgG1f and IgG1f-S267Erespectively, despite containing the CH1 and hinge regions of an A-formIgG2 antibody (IgG2.3) (Table 21). Similar data was also obtained usingIgG2.5G1-AY and IgG2.5G1-AY-V27 antibodies, demonstrating the successfulengineering of B-form IgG2 antibodies (containing C131S mutation termedIgG2.5) having IgG1f or modified IgG1f like effector functions. Data forseveral other antibodies with IgG2.3G1-AY, IgG2.3G1-AY-V27, IgG2.5G1-AY,or IgG2.5G1-AY-V27 constant regions but different variable regions showsthat this engineering strategy is broadly applicable to other antibodiesindependent of the variable domains (Table 21).

TABLE 21 % Rmax values for 1 uM antibodies binding to anti-his Fabcaptured FcgR-his proteins hCD32a- hCD32a- hCD16a- hCD16B- mAb hCD64H131 R131 hCD32b V158 NA2 mAb8-IgG1f 80%  82% 51% 27%  51%  21% mAb9-IgG1f 70%  33% 19% 4% 28%  10%  mAb11-IgG2.3 2% 44% 17% 5% 1% 0%mAb6-IgG2.3 3% 66% 14% 3% 1% 0% mAb4-IgG2.3 1% 39%  6% 1% 1% 0%mAb5-IgG2.3 6% 100%  30% 4% 3% 0% mAb12-IgG2.3 2% 39%  7% 1% 1% 0%mAb13-IgG2.3 2% 40%  7% 1% 1% 0% mAb11-IgG2.5 0% 40% 13% 3% 0% −1% mAb7-IgG2.5 4% 72% 19% 2% 2% 0% mAb8-IgG2.5 3% 59% 14% 3% 2% 0%mAb10-IgG2.5 1% 29%  5% 1% 1% 0% mAb6-IgG2.5 3% 75% 17% 4% 2% 0%mAb4-IgG2.5 2% 46%  8% 1% 1% 0% mAb5-IgG2.5 6% 89% 26% 5% 4% 1%mAb12-IgG2.5 1% 36%  6% 1% 1% 0% mAb13-IgG2.5 −2%  39%  4% −2%  0% −2% mAb8-IgG2.3G1-AY 77%  61% 38% 10%  38%  13%  mAb10-IgG2.3G1-AY 67%  23%14% 4% 24%  8% mAb7-IgG2.5G1-AY 80%  73% 45% 12%  47%  19% mAb8-IgG2.5G1-AY 77%  70% 45% 17%  48%  22%  mAb7-IgG2.3G1-AY-V27 84% 68% 92% 76%  26%  7% mAb8-IgG2.3G1-AY-V27 78%  67% 80% 67%  24%  7%mAb10-IgG2.3G1-AY-V27 69%  24% 57% 40%  12%  3% mAb7-IgG2.5G1-AY-V2781%  74% 89% 84%  32%  9% mAb8-IgG2.5G1-AY-V27 77%  76% 79% 77%  33% 10% 

Example 27: Effects of Anti-OX40 Antibodies with Modified Heavy ChainConstant Regions on T Cell Proliferation and IFN-γ and IL-2 Secretionfrom T Cells with or without Cross-Linking

Anti-OX40 antibodies with modified IgG2 CH1/hinge regions may have theability to promote T cell activation in the absence of cross-linking,and thus may be able to promote T cell activation in vivo in the absenceor low expression of cell types expressing FcγRs, and possibly topromote anti-tumor activity in a wider range of tumor types than IgG1isotype antibodies.

Alternatively, modified CH1/hinge region antibodies may still requirecross-linking in order to promote T cell activation, but may haveincreased agonist activity when bound to FcγRs compared to IgG1 isotypeantibodies, and thus be more potent in promoting T cell activation andanti-tumor activity.

Anti-OX40 antibodies having modified heavy chain constant regionscomprising the sequences shown in Table 22 are generated and tested fortheir effects on T cell proliferation and IFN-γ and IL-2 secretion fromT cells with or without cross-linking using the assays described below.The light chain sequences for antibodies OX40.6, 40.8, 40.16, and 40.21correspond to SEQ ID NOs: 96, 110, and 116 (for both OX40.16 and 40.21),respectively.

TABLE 22 Constructs SEQ ID NO OX40.6-Vh-hHC-IgG2.3 282OX40.8-Vh-hHC-IgG2.3 283 OX40.16-Vh-hHC-IgG2.3 284OX40.6-Vh-hHC-IgG2.3G1 285 OX40.8-Vh-hHC-IgG2.3G1 286OX40.16-Vh-hHC-IgG2.3G1 287 OX40.6-Vh-hHC-IgG2.3G1-V27 288OX40.8-Vh-hHC-IgG2.3G1-V27 289 OX40.16-Vh-hHC-IgG2.3G1-V27 290OX40.6-Vh-hHC-IgG2.5 291 OX40.8-Vh-hHC-IgG2.5 292 OX40.16-Vh-hHC-IgG2.5293 OX40.21-Vh-hHC-IgG2.5 294 OX40.21-Vh-hHC-IgG2.5G1 295OX40.21-Vh-hHC-IgG2.5G1-V27 296CHO-CD3+/−CD32 assay

Anti-OX-40 antibodies with the sequences shown in Table 22 are testedfor their ability to induce T cell activity in vitro by measuring theproliferation of and amount of IL-2 and IFN-γ secreted by T cellsincubated with the antibodies.

Transfected CHO cell lines are generated for use as artificialantigen-presenting cells in a primary T cell activation assay. TheCHO-CD3-CD32A cell line expresses anti-human CD3 antibody in asingle-chain Fv format, along with the human Fc receptor (FcR) CD32A topresent anti-OX40 antibodies on the CHO cell surface. The CHO-CD3 cellline expresses anti-human CD3 antibody in a single-chain Fv formatwithout FcR.

Briefly, human primary CD4 T cells are isolated by negative selection(RosetteSep™, StemCell Technologies) and co-cultured with eitherirradiated CHO-CD3-CD32A cells, or irradiated CHO-CD3 cells, at an 8:1T:CHO ratio, in the presence of graded doses of anti-OX40 antibodies orisotype control antibody. After 3 to 4 days in culture at 37° C.,supernatants are harvested for assessment of T cell activation by meansof measurement of secreted human IFN-γ either by ELISA (BD Biosciences)or HTRF assay (Cisbio), following the manufacturers' recommendations.Afterwards, tritiated thymidine is added for the final approximately 18hours of culture to measure T proliferation by tritiated thymidineincorporation, as an additional assessment of T cell activation.

SEB PBMC Assay

Anti-OX-40 antibodies with the sequences shown in Table 22 are testedfor their effects on stimulating primary T cells in cultures ofstaphyloccus enterotoxin B (SEB)-activated human peripheral bloodmononuclear cells (PBMCs). Human whole blood samples are obtained fromAllCells, Inc. (Berkeley, Calif.) or from donors at Bristol-MyersSquibb, Redwood City, Calif. under the auspices of an in-housephlebotomy program. PBMCs are isolated by gradient purification on aFicoll-Hypaque cushion and cultured for 3 days in culture mediumsupplemented with fixed, suboptimal (85 ng/mL) of superantigenstaphylococcus enterotoxin B (SEB; Toxin Technologies, Sarasota, Fla.)in the presence of graded doses of OX40 antibodies or isotype controlantibody. In some cases, 2-5 μg/mL of soluble cross-linking antibody,F(ab′)2 goat anti-human Fcγ, is also added to the cultures. Afterculturing for 3 days at 37° C., supernatants are harvested forassessment of T cell activation by means of ELISA measurement ofsecreted human IL-2. Briefly, culture supernatants are diluted 1:10 insample diluent and tested for the presence of human IL-2 by ELISA (BDBioscience) per the manufacturer's recommended protocol. Following theaddition of TMB substrate, assay plates are read on a Spectramax 340PCreader using Softmax operating software at a wavelength of 650 nm.Measured optical densities of the chromogenic substrate wereproportional to bound detecting antibody.

MLR Assay

Anti-OX-40 antibodies with the sequences shown in Table 22 tested fortheir ability to potentiate primary human T cell proliferation andIFN-γsecretion in a T cell: Dendritic Cell Allogeneic Mixed LymphocyteReaction (T:DC AlloMLR). Total T cells are isolated from peripheralblood from healthy human donors by negative selection (RosetteSep,Stemcell Technologies). Monocytes are isolated from peripheral bloodfrom healthy human donors using CD14 microbeads (Miltenyi), and culturedfor six days in the presence of GM-CSF and IL-4 to derive immaturedendritic cells (DCs). DCs and T cells are co-cultured in the presenceof graded doses of OX40 antibodies or isotype control antibody. In somecases, 2-5 μg/mL of soluble cross-linking antibody, such as F(ab′)2 goatanti-human Fcγ, is also added to the cultures. Supernatant from eachsample is harvested between Day 4 and Day 7 for measurement of secretedIFN-γ by ELISA (BD Biosciences) or HTRF assay (Cisbio), following themanufacturers' recommendations. After supernatant harvest, the cellcultures are pulsed with 1 μCi/well of ³[H]-thymidine for the last 16-18hours of the culture. The cells are harvested onto filter plates, and³[H] counts per minute of cell-incorporated ³[H]-thymidine are read as ameasure of T cell proliferation.

Example 28: Anti-Tumor Activity of OX40 Agonist mAb with CTLA-4 Blockadein a CT26 Model

To test for synergy between anti-OX-40 antibody and anti-CTLA-4 antibodytreatments, combinations of these antibodies were tested in the CT26mouse tumor model. Mice were inoculated with CT26 tumors and mAb dosingwas initiated at Day +3 following inoculation (dosed on Days 3, 7, and10) with 200 μg/mouse of the antibodies indicated in FIGS. 41A-41D.

As shown in FIGS. 41A-41D, both the anti-CTLA-4 antibody (FIG. 41B) andanti-OX40 antibody (FIG. 41C) showed minimal activity when used alone (1of 8 mice tumor free for both treatments), but had significantanti-tumor activity when combined (FIG. 41D), with 4 of 8 mice renderedtumor free.

Example 29: Phase 1/2a Trial in Subject Having Solid Tumors

A Phase 1/2a study of OX40.21 administered alone or in combination withnivolumab or ipilimumab is conducted in subjects having advanced solidtumors to demonstrate the efficacy of administering OX40.21 alone or incombination with nivolumab or ipilimumab.

1. Objective

The primary objective of the study is to assess the safety,tolerability, dose-limiting toxicities (DLTs), and maximum tolerateddose (MTD)/recommended phase 2 dose (RP2D) of OX40.21 administered aloneor in combination with nivolumab or ipilimumab in subjects with advancedmalignant tumors.

Secondary objectives include investigating the preliminary anti-tumoractivity of OX40.21 administered alone or in combination with nivolumabor ipilimumab in subjects with advanced malignant tumors; characterizingthe PK of OX40.21 administered alone and in combination with nivolumabor ipilimumab; and characterizing the immunogenicity of OX40.21administered alone or in combination with nivolumab or ipilimumab andthe immunogenicity of nivolumab or ipilimumab administered with OX40.21.Additional exploratory objectives include exploring potentialassociations between anti-tumor activity and select biomarker measuresin tumor biopsy specimens and peripheral blood prior to treatment andfollowing administration of OX40.21 alone or in combination withnivolumab or ipilimumab; assessing the potential effect of OX40.21monotherapy and combination therapy on QTc interval; characterizingnivolumab PK in subjects receiving the combination of nivolumab andOX40.21; characterizing ipilimumab PK in subjects receiving thecombination of ipilimumab and OX40.21; assessing the overall survival(OS) in subjects treated with OX40.21 alone and in combination withnivolumab or ipilimumab; and exploring potential relationships betweendose/exposure and anti-tumor activity, pharmacodynamic (PD) effects(selected biomarkers in the peripheral blood and tumor biopsyspecimens), and key safety measures in subjects treated with OX40.21alone and in combination with nivolumab or ipilimumab.

2. Study Design and Duration

This is a Phase 1/2a, open label study of OX40.21 in subjects withadvanced solid tumors that integrates initial OX40.21 monothereapy withsubsequent nivolumab or ipilimumab combination therapy.

Study sections (dose escalation and dose expansion) proceed in a phasedapproach based on study-emergent safety, PK, and PD data. The firstsection of the study begins with OX40.21 monotherapy dose escalationcohorts. Clinical data from the first 3 monotherapy dose cohorts serveas a foundation for initiating dose escalation of OX40.21 in combinationwith nivolumab. Clinical data from the first 3 monotherapy dose cohortsin addition to the clinical data from the first cohort of OX40.21 incombination with nivolumab serve as a foundation for initiating doseescalation of OX40.21 in combination with ipilimumab. Afterestablishment of a tolerable and pharmacologically active RP2D ofOX40.21 in the dose escalation section, dose expansion in specific tumorcohorts is initiated.

3. Dose Escalation

A schematic of the study design for Part 1A is shown in FIG. 42 .

The dose escalation phase of the study evaluates the safety andtolerability of OX40.21, alone or in combination with nivolumab oripilimumab, in subjects with advanced solid tumors.

The initial dose level of OX40.21 is 20 mg. Dose escalation decisionsfor subsequent doses are based on DLTs using a BLRM model (for OX40.21monotherapy) or a BLRM (−Copula) model (for OX40.21 in combination withnivolumab or ipilimumab). The DLT period is 28 days for both monotherapyand combination therapy dose escalation parts. The DLT rate isdetermined based on the incidence, severity, and duration of AEs thatoccur within the DLT period and for which no alternative cause can beidentified. Dose selection for the next monotherapy cohort/dose leveltakes into account the BLRM (−Copula) recommendation in conjunction withall available PK, PD, and clinical and laboratory safety data from alltreated subjects. Starting dose selection of OX40.21 for Part 2A isdetermined using data available from Part 1A, including clinical andlaboratory safety assessments, PK/PD data, and modeling recommendationwithin Bayesian hierarchical modeling framework by incorporatingsingle-agent toxicity profiles of both OX40.21 (Part 1A) and nivolumab(CA209-003). Starting dose selection of OX40.21 for Part 3A isdetermined using data available from Parts 1A and 2A, including clinicaland laboratory safety assessments, PK/PD data, and modelingrecommendation within Bayesian modeling framework by incorporatingsingle-agent toxicity profiles of both OX40.21 (Part 1A) and ipilimumab(CA184-022). Actual doses can be modified per the BLRM (−Copula), but donot exceed doubling of the previously tested dose.

During dose escalation for all dose cohorts, the initial subject(sentinel subject) is observed for 5 days before additional subjects inthat cohort are treated with study drug.

Approximately 30 subjects are enrolled in each dose escalation part. Thenumber of subjects in each dose escalation cohort varies depending onBLRM (−Copula) recommendations. Initially, approximately 3 subjects aretreated at the starting dose levels of OX40.21 or OX40.21 in combinationwith nivolumab or ipilimumab. Additional cohorts of approximately 3evaluable subjects are treated at recommended dose levels per BLRM(−Copula) during the dose escalation phase. At least 6 DLT-evaluablesubjects are treated at the MTD.

Part 1A: Enrollment begins in Part 1A, OX40.21 monotherapy doseescalation. The initial dose of OX40.21 for Part 1A is 20 mg, withexpected subsequent doses of 40, 80, 160, and 320 mg. Actual doses canbe modified per the BLRM but do not exceed doubling of the previouslytested dose.

Part 2A: Part 2A is the combination arm of OX40.21 with nivolumab thatis initiated after at least 3 dose levels in the monotherapy doseescalation are found to be tolerated or an MTD has been determined inthe monotherapy dose escalation (Part 1A). The starting dose of OX40.21in Part 2A is at least 1 dose level below a dose demonstrated to betolerated in Part 1A to ensure further safety of the combination. At notime does the dose for OX40.21 in Part 2A exceed the highest tolerateddose in Part 1A. Nivolumab is administered at a flat dose of 240 mg.Each treatment cycle is 2 weeks in length and study drugs areadministered every 2 weeks starting on Day 1 of each cycle for up to 12cycles.

Part 3A: Part 3A is the combination arm of OX40.21 with ipilimumab thatis initiated only after at least 3 dose levels in the monotherapy doseescalation are found to be tolerated or an MTD is determined in themonotherapy dose escalation (Part 1A) and at least 1 dose cohort isfound to be tolerated in the OX40.21 with nivolumab dose escalationpart. The starting dose of OX40.21 in Part 3A is at least 1 dose levelbelow a dose demonstrated to be tolerated in Part 1A. At no time doesthe dose for OX40.21 in Part 3A exceed the highest tolerated dose inPart 1A to further ensure safety of the combination doses in treatedsubjects. Ipilimumab is administered at a dose of 1 mg/kg. Eachtreatment cycle is 3 weeks in length. OX40.21 is administered every 3weeks starting on Cycle 1 Day 1, up to and including 8 cycles, andipilimumab is administered every 3 weeks starting on Day 1 for 4 cycles.Only OX40.21 is administered in the last 4 cycles.

Dose Expansion:

Treatment in the dose expansion cohorts is initiated when the MTD/RP2Dhas been determined based on the evaluation of totality of availableclinical safety (DLTs, significant AEs occurring after the DLT period),PK, PD, and modeling data from the dose escalation (Parts 1A, 2A, and3A). Approximately 110 subjects are treated in all dose expansioncohorts.

Part 1B is the OX40.21 monotherapy dose expansion cohort in subjectswith cervical cancer at the MTD/RP2D determined in Part 1A. Dosing ofOX40.21 begins on Day 1 of each cycle and is administered every 2 weeksfor up to 12 cycles. Approximately 12 subjects are treated in thisexpansion cohort.

Parts 2B is the combination therapy (OX40.21 with nivolumab) doseexpansion part in subjects with CRC at the MTD/RP2D determined in Part2A. Nivolumab is administered at a flat dose of 240 mg. Each treatmentcycle is 2 weeks in length and study drugs are administered every 2weeks starting on Day 1 of each cycle for up to 12 cycles. Approximately35 subjects are treated in this expansion cohort.

Part 2C is the combination therapy (OX40.21 with nivolumab) doseexpansion part in subjects with BC at the MTD/RP2D determined in Part2A. Each treatment cycle is 2 weeks in length and study drugs areadministered every 2 weeks starting on Day 1 of each cycle for up to 12cycles. Approximately 27 subjects are treated in this expansion cohort.

Part 3B is the combination therapy (OX40.21 with ipilimumab) doseexpansion part in subjects with OC at the MTD/RP2D determined in Part3A. Each treatment cycle is 3 weeks in length. Ipilimumab isadministered in the initial 4 cycles in combination with OX40.21. Thenthe subject continues on OX40.21 monotherapy for up to an additional 4cycles for a total of up to 24 weeks (8 cycles) of treatment.Approximately 35 subjects with OC are treated in this expansion cohort.

Summary of Study Periods:

Subjects complete up to 5 periods in the study: Screening (up to 28days), Treatment (up to 24 weeks), Safety Follow-up (minimum 100 days),Response Follow-up, and Survival Long-term Follow-up (up toapproximately 2 years from the first dose) as described below. The studyvisit schematic is presented in FIG. 43 .

Screening Period:

The Screening period lasts for up to 28 days. The screening periodbegins by establishing the subject's initial eligibility and signing ofthe informed consent form. Subjects are enrolled using an InteractiveResponse Technology (IRT).

Treatment Period:

The Treatment period consists of up to 24 weeks of dosing. Followingeach treatment cycle, the decision to treat a subject with the nextcycle of study therapy, up to 24 weeks of treatment, is based onrisk/benefit and tumor assessments. Tumor assessments are performedevery 8 weeks for every 2-week (q2w) dosing regimen and every 9 weeksfor every 3-week (q3w) dosing regimen. Assessments of partial response(PR) and complete response (CR) mare confirmed at least 4 weeksfollowing initial assessment. Tumor progression or response endpointsare assessed using Response Evaluation Criteria In Solid Tumors (RECIST)v1.1.

Subjects with a response of stable disease (SD), PR, or CR at the end ofa given cycle continue to the next treatment cycle. Subjects aregenerally allowed to continue study therapy until the first occurrenceof one of the following: 1) completion of the maximum number of cycles;2) progressive disease; 3) clinical deterioration suggesting that nofurther benefit from treatment is likely; 4) intolerability to therapy;or 5) meeting the criteria for discontinuation of study therapy.

Safety Follow-Up:

Upon completion of study therapy, subjects enter the Safety Follow-upperiod. After the end of treatment (EOT) visit, subjects are evaluatedfor any new adverse events (AEs) for at least 100 days after the lastdose of therapy. Follow-up visits occur at Days 30, 60 and 100 after thelast dose or the date of discontinuation. Subjects (except those whowithdraw consent for study participation) complete 3 clinical SafetyFollow-up visits regardless of whether they start new anti-cancertherapy.

Survival Follow-Up:

After completion of the Safety Follow-up period, subjects enter theSurvival Follow-up period. Subjects are followed approximately every 3months (12 weeks) until death, lost to follow-up, withdrawal of consent,or conclusion of the study, whichever comes first. The duration of thisphase is up to 2 years following the first dose of study drug.

Response Follow-Up:

After completion of the Safety Follow-up period, all subjects withongoing SD, PR, or CR at the EOT visit enter the Response Follow-upperiod, which occurs simultaneously with the Survival Follow-up period.These subjects continue to have radiological and clinical tumorassessments every 3 months (12 weeks) during the Response Follow-upperiod or until disease progression or withdrawal of study consent.Radiological tumor assessments for subjects who have ongoing clinicalbenefit continue to be collected after subjects complete the survivalphase of the study. Subjects who have disease progression followinginitial course of study therapy are not evaluated for response beyondthe EOT visit and are allowed to receive other tumor directed therapy asrequired.

Duration of Study:

The total duration of study time for any individual subject isapproximately 2 years. The study ends when the last subject completestheir last study visit, which is approximately 4 years after the startof the study.

Number of Subjects:

Approximately 225 subjects will be enrolled, and approximately 200subjects will be treated in the study.

Study Population:

Subjects are at least 18 years old and have histologic or cytologicconfirmation of a malignancy that is advanced (metastatic, recurrent,refractory and/or unresectable) with measurable disease per RECIST v1.1.

Dose Escalation and Stopping Rules

In Parts 1A, 2A, and 3A, the BLRM and BLRM (−Copula) models are utilizedfor dose escalation recommendations after DLT information becomesavailable for each cohort of subjects. OX40.21 dose selection for thenext cohort/dose level takes into account the BLRM (−Copula)recommendation in conjunction with clinical recommendation and allavailable PK, PD, and clinical and laboratory safety data from alltreated subjects.

Dose-Limiting Toxicities

To guide dose escalation, DLTs are defined based on the incidence,intensity, and duration of AEs for which no clear alternative cause isidentified. The DLT period is 28 days of initiation of the studydrug(s). For subject management, an AE that meets DLT criteria,regardless of the cycle in which it occurs, leads to discontinuation ofstudy drug. Subjects who withdraw from the study during the DLTevaluation interval for reasons other than a DLT may be replaced with anew subject at the same dose level. The incidence of DLT(s) during theDLT evaluation period is used in dose escalation decisions and to definethe MTD. AEs occurring after the DLT period are considered for thepurposes of defining the MTD, if they are determined to have no clearalternative cause and are not related to disease progression. Subjectsexperiencing a DLT are not retreated with study drug and enter thesafety follow-up period of the study. AEs are graded according to theNational Cancer Institute (NCI) Common Terminology Criteria for AdverseEvents (CTCAE) v4.03.

Non-Hematologic DLT:

A. Hepatic DLT

-   -   Any ≥Grade 3 elevation of AST, ALT, or total bilirubin    -   AST, ALT, or total bilirubin,    -   Grade 2 AST or ALT with symptomatic liver inflammation (e.g.,        right upper quadrant tenderness, jaundice, pruritis)    -   AST or ALT >3×ULN and concurrent total bilirubin >2×ULN without        initial findings of cholestasis (elevated serum alkaline        phosphatase [ALP]) (e.g., findings consistent with Hy's law or        FDA definition of potential drug-induced liver injury or pDILI)

B. Non-hepatic DLT

-   -   Grade 2 or greater uveitis, episcleritis, or iritis    -   Any other Grade 2 eye pain or blurred vision that does not        respond to        topical therapy and does not improve to Grade 1 severity within        2 weeks OR requires systemic treatment    -   Grade 3 or greater pneumonitis, bronchospasm, neurologic        toxicity, hypersensitivity reaction, or infusion reaction    -   Any Grade 3 or greater non-dermatologic, non-hepatic toxicity        will be considered a DLT with the following specific exceptions:    -   Grade 3 or Grade 4 electrolyte abnormalities that are not        complicated by associated clinical adverse experiences, last        less than 72 hours, and either resolve spontaneously or respond        to conventional medical intervention    -   Grade 3 nausea, vomiting, or diarrhea that lasts less than 72        hours, and either resolves spontaneously or responds to        conventional medical intervention    -   Grade 3 or 4 elevation of amylase or lipase not associated with        clinical or radiographic evidence of pancreatitis    -   Isolated Grade 3 fever not associated with hemodynamic        compromise (e.g., hypotension, clinical, or laboratory evidence        of impaired end-organ perfusion)    -   Grade 3 endocrinopathy that is well controlled by hormone        replacement    -   Grade 3 tumor flare (defined as pain, irritation, or rash that        localizes to sites of known or suspected tumor)    -   Grade 3 fatigue for less than 7 days    -   Grade 3 infusion reaction that returns to Grade 1 in less than 6        hours

Dermatologic DLT

-   -   Grade 4 rash    -   Grade 3 rash if no improvement (i.e., resolution to 5 Grade 1)        after a 1- to 2-week infusion delay. Subjects who have not        experienced a Grade 3 skin AE may resume treatment in the        presence of Grade 2 skin toxicity.

Hematologic DLT

-   -   Grade 4 neutropenia ≥5 days in duration    -   Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with        clinically significant bleeding, or any requirement for platelet        transfusion    -   Grade 4 anemia not explained by underlying disease    -   Grade 4 febrile neutropenia    -   Grade 3 febrile neutropenia that lasts >48 hours    -   Grade ≥3 hemolysis (i.e., requiring transfusion or medical        intervention such as steroids)        Treatment with Additional Cycles Beyond 24 Weeks

Subjects are treated for 24 weeks unless criteria for study drugdiscontinuation are met earlier. Subjects completing approximately 24weeks of treatment with ongoing disease control (CR, PR, or SD) areeligible for an additional 24 weeks of study therapy in monotherapy(Part 1) and combination therapy (Parts 2 and 3) beyond the initial 24weeks when the risk/benefit assessment favors continued administrationof study therapy. Upon completion of the additional 24 weeks of studytherapy, subjects enter the Safety Follow-up period.

Treatment Beyond Progression

Treatment beyond progression is allowed in select subjects with initialRECIST v1.1-defined progressive disease after determining that thebenefit/risk assessment favors continued administration of study therapy(e.g., subjects are continuing to experience clinical benefit,tolerating treatment, and meeting other criteria).

Retreatment

Retreatment is allowed if confirmed disease progression occurs duringthe response follow-up period. Subjects completing approximately 24weeks (or additional cycles of treatment, if appropriate) of therapy whoenter the response follow-up period with ongoing disease control (CR,PR, or SD) without any significant toxicity are eligible forretreatment. Such subjects are eligible for retreatment on acase-by-case basis after evaluation and determining whether therisk/benefit ratio supports administration of further study therapy, andthe subject continues to meet eligibility criteria for treatment withstudy therapy. Subjects meeting criteria for retreatment are treatedwith the originally assigned monotherapy or combination therapy regimen(e.g., the same dose and dose schedule as administered during the first24 weeks), unless that dose and schedule were subsequently found toexceed the MTD, in which case the subject is treated at the next lowerdose deemed tolerable/safe.

Inclusion Criteria

1) Signed Written Informed Consent

-   -   a) The subject must sign the informed consent form prior to the        performance of any study-related procedures that are not        considered part of SOC.    -   b) Consent for tumor biopsy samples (mandatory pre- and        on-treatment biopsies are required for the dose expansion        cohorts, and for additional subjects added to any of the        previously completed dose escalation cohorts and optional for        dose escalation cohorts).

2) Target Population

-   -   Subjects must be at least 18 years old and have histologic or        cytologic confirmation of a malignancy that is advanced        (metastatic, recurrent, refractory, and/or unresectable) with        measurable disease per RECIST v1.1.    -   A. Dose Escalation:        -   Subjects must have received, and then progressed, or have            been refractory or intolerant to, at least 1 standard            treatment regimen in the advanced or metastatic setting, if            such a therapy exists. Subjects who are ineligible for any            standard therapy are allowed to enroll provided their            ineligibility is documented in medical records. The            following tumor histologies are permitted except for            subjects with primary central nervous system (CNS) tumors,            or with CNS metastases as the only site of active disease.    -   (i) Melanoma: BRAF mutation status must be documented if known.    -   (ii) NSCLC: EGFR, ALK, KRAS, and ROS1 mutational status must be        documented if known    -   (iii) Head and neck cancer restricted to squamous cell        carcinoma. HPV status must be documented if known    -   (iv) Transitional cell carcinoma of the genitourinary tract    -   (v) Renal cell carcinoma    -   (vi) Pancreatic adenocarcinoma    -   (vii) CRC: MSI, KRAS, and BRAF status must be documented if        known.    -   (viii) Cervical cancer: HPV status must be documented if known.    -   (ix) Triple negative breast cancer HER2, ER and PR status must        be documented    -   (x) Adenocarcinoma of the endometrium    -   (xi) Ovarian cancer    -   (xii) Prostate adenocarcinoma    -   (xiii) Hepatocellular cancer-Child Pugh A only    -   (xiv) Small cell lung cancer    -   (xv) Gastric and gastric esophageal junction cancer: HER2 Status        must be documented if known.    -   B. Dose Expansion: Parts 1B, 2B, 2C, and 3B    -   The following tumor types will be permitted:        -   (a) Cervical Cancer—Part 1B            -   (i) Histologically confirmed cervical cancer that is                unresectable, metastatic, or recurrent with documented                disease progression            -   (ii) Document tumor HPV status if known. If unknown,                subjects must consent to allow their submitted archived                tumor tissue sample (block or unstained slides) to be                tested.            -   (iii) Prior therapy requirement:    -   1. Must have received and then progressed or have been        intolerant or refractory to at least 1 standard systemic        therapy, for metastatic and/or unresectable disease (e.g.,        paclitaxel/cisplatin, paclitaxel/cisplatin/bevacizumab).        Concurrent chemotherapy administered with primary radiation and        adjuvant chemotherapy given following completion of radiation        therapy do not count as systemic chemotherapy regimens.        -   (b) Colorectal Cancer—Part 2B            -   (i) Histologically confirmed CRC that is metastatic or                recurrent with documented disease progression            -   (ii) Document MSI, MMR, KRAS, and BRAF status if known.                If unknown, subjects must consent to allow their                submitted archived tumor tissue sample (block or                unstained slides) to be tested.            -   (iii) Prior therapy requirement:                -   Subjects must have received and then preogressed or                    have been intolerant or refractory to at least 1                    standard systemic therapy, for metastatic and/or                    unresectable disease (or have progressed within 6                    months of adjuvant therapy).        -   (c) Bladder Cancer—Part 2C            -   (i) Histologically or cytologically confirmed urothelial                carcinoma (including mixed histologies of urothelial                carcinoma with elements of other subtypes) of the renal                pelvis, ureter, bladder, or urethra with progression or                refractory disease            -   (ii) Prior therapy requirement:                -   Subjects must have received and then progressed or                    have been intolerant or refractory to at least 1                    standard systemic therapy (e.g., platinum based                    chemotherapy) regimen for the treatment of                    metastatic (Stage IV) or locally advanced                    unresectable disease.        -   (d) Ovarian—Part 3B            -   (i) Histologically or cytologically confirmed ovarian                carcinoma (including epithelial OC, primary peritoneal,                or fallopian tube carcinoma) with documented disease                progression            -   (ii) Documented germline BRCA mutation status, if known.                If unknown, subjects must consent to allow their                submitted archived tumor tissue sample (block or                unstained slides) to be tested.            -   (iii) Prior therapy requirement:                -   Subjects must have received and then progressed or                    have been intolerant or refractory to at least 1                    standard systemic therapy (e.g., platinum-based                    chemotherapy), for metastatic and/or unresectable                    disease.

3) Eastern Cooperative Oncology Group (ECOG) performance status of ≤1.

4) Presence of at least 1 lesion with measurable disease as defined byRECIST v1.1 for response assessment. Subjects with lesions in apreviously irradiated field as the sole site of measurable disease arepermitted to enroll provided the lesion(s) have demonstrated clearprogression and can be measured accurately.

5) For subjects requiring fresh tumor biopsy, subjects must have atleast one lesion accessible for pre- and on-treatment biopsy, inaddition to the minimum one RECIST v1.1 measureable lesion required forresponse assessment. This lesion needs to be distinct from indexlesion(s) being evaluated for radiological response.

6) Subjects with prior exposure to therapy with any agent specificallytargeting checkpoint pathway inhibition (such as anti-PD-1, anti-PD-L1,anti-PD-L2, anti-LAG-3, and anti-CTLA-4 antibody) are permitted after awashout period of any time greater than 4 weeks from the last treatment

-   -   Note: (i) Subjects who experienced prior Grade 1 to 2 checkpoint        therapy-related immune-mediated AEs must have confirmed recovery        from these events at the time of study entry, other than        endocrinopathies treated with supplementation, as documented by        resolution of all related clinical symptoms, abnormal findings        on physical examination, and/or associated laboratory        abnormalities. Where applicable, these subjects must also have        completed steroid tapers for treatment of these AEs by a minimum        of 14 days prior to commencing treatment with study        therapy. (ii) Eligibility of subjects with prior ≥Grade 3        checkpoint therapy-related immune AEs, will be considered on a        case-by-case basis after discussion with the Medical Monitor        (e.g., asymptomatic isolated Grade 3 lipase elevations without        clinical or radiological features of pancreatitis are permitted        to enroll).

7) Subjects with prior therapy with any agent specifically targetingT-cell co-stimulation pathways except anti-OX40 antibody, anti-CD137,anti-GITR antibody, and anti-CD27 are permitted after a washout periodof any time greater than 4 weeks from the last treatment.

8) Prior palliative radiotherapy must have been completed at least 2weeks prior to first dose of study drug. Subjects with symptomatic tumorlesions at baseline that may require palliative radiotherapy within 4weeks of first dose of study drug are strongly encouraged to receivepalliative radiotherapy prior to enrollment.

9) Subjects enrolled into dose escalation and expansion cohorts mustconsent to the acquisition of existing formalin-fixed, paraffin-embedded(FFPE) tumor tissue, either a block or a minimum of 15 unstained slides(25 slides preferred), for performance of correlative studies. If anarchived sample is not available, subject must consent to apre-treatment tumor biopsy. Subjects unable to provide an archived tumorsample and who either do not consent to a pre-treatment tumor biopsy ordo not have accessible lesions are not eligible. (However, subjectswhose pre-treatment biopsy yields inadequate tissue quantity or qualitywill not be ineligible on this basis alone). For any additional subjectsadded to any of the previously completed dose escalation cohorts,mandatory pre- and on-treatment biopsies are required.

10) Subjects enrolled into dose expansion, or added to any previouslycompleted dose escalation cohort, are required to undergo mandatory pre-and ontreatment biopsies at acceptable clinical risk. (a) The solidtumor tissue specimen must be a core needle, excisional, or incisionalbiopsy. Fine needle biopsies, drainage of pleural effusions withcytospins, or punch biopsies are not considered adequate for biomarkerreview. Biopsies of bone lesions that do not have a soft tissuecomponent or decalcified bone tumor samples are also not acceptable. (b)Biopsied lesions should be distinct from index lesion(s) being evaluatedfor radiological response

11) Adequate organ function for subjects as defined by the following:

-   -   (a) Neutrophils ≥1500/μL (stable off any growth factor within 4        weeks of first study drug administration)    -   (b) Platelets ≥80×103/μL (transfusion to achieve this level is        not permitted within 2 weeks of first study drug administration)    -   (c) Hemoglobin ≥8 g/dL (transfusion to achieve this level is not        permitted within 2 weeks of first study drug administration)    -   (d) ALT and AST ≤3×upper limit of normal (ULN)    -   (e) Total bilirubin ≤1.5×ULN (except subjects with Gilbert's        Syndrome who must have normal direct bilirubin)    -   (f) Normal thyroid function or stable on hormone supplementation        per investigator assessment    -   (g) Albumin ≥2 mg/dl    -   (h) Serum creatinine ≤1.5×ULN or creatinine clearance (CrCl) ≥40        ml/min (measured using the Cockcroft-Gault formula below):

Female CrCl=(140−age in years)×weight in kg×0.85 72×serum creatinine inmg/dL

Male CrCl=(140−age in years)×weight in kg×1.00 72×serum creatinine inmg/dL

12) Ability to comply with treatment, PK and PD sample collection, andrequired study follow-up

Age and Reproductive Status

a) Men and women, ages ≥18 years at the time of informed consent.

b) Women of childbearing potential (WOCBP) must have a negative serum orurine pregnancy test (minimum sensitivity 25

IU/L or equivalent units of human chorionic gonadotrophin [hCG]) within24 hours prior to the start of study drug.

c) Women must not be breastfeeding.

d) WOCBP must agree to follow instructions for method(s) ofcontraception for the duration of treatment with study drug OX40.21 plus5 half-lives of study drug plus 30 days. This duration should be 12weeks for Parts 1 and 3 subjects (50 days plus 30 days) or 23 weeks forPart 2 subjects (130 days plus 30 days [duration of ovulatory cycle]),for a total of up to 160 days post-treatment completion.

e) Men who are sexually active with WOCBP must agree to followinstructions for method(s) of contraception for the duration oftreatment with study drug OX40.21 plus 5 half-lives of the study drugplus 90 days. The duration should be 20 weeks for Parts 1 and 3 subjects(50 days plus 90 days) or 31 weeks for Part 2 subjects (130 dayscompletion. In addition, male subjects must be willing to refrain fromsperm donation during this time.

f) Azoospermic males are exempt from contraceptive requirements. WOCBPwho are continuously not heterosexually active are also exempt fromcontraceptive requirements, but still undergo pregnancy testing.

Exclusion Criteria 1) Target Disease Exceptions

a) Subjects with known or suspected CNS metastases or untreated CNSmetastases, or with the CNS as the only site of disease, are excluded.However, subjects with controlled brain metastases are allowed toenroll. Controlled brain metastases are defined as no radiographicprogression for at least 4 weeks following radiation and/or surgicaltreatment (or 4 weeks of observation if no intervention is clinicallyindicated), and off of steroids for at least 2 weeks, and no new orprogressive neurological signs and symptoms.

b) Subjects with carcinomatous meningitis

c) For ovarian cancer:

-   -   i) ovarian cancer subjects with history of bowel obstruction in        the prior 6 months or with Tenckhoff catheter are excluded.    -   ii) up to 4 prior anti-cancer treatments are permitted (i.e,        chemotherapy, radiotherapy, hormonal, or immunotherapy).        Restarting the same regimen after a drug holiday may be        considered one regimen; however it would be counted as two        regimens if there was any other regimen used in between.

2) Medical History and Concurrent Diseases

a) Subjects with a prior malignancy, different from the one used forenrollment in this study, diagnosed within less than 2 years prior tostudy entry are excluded (except non-melanoma skin cancers and in situ

cancers such as bladder, colon, cervical/dysplasia, melanoma, orbreast). In addition, subjects with other second malignancies diagnosedmore than 2 years ago who have received therapy with curative intentwith no evidence of disease during the interval who are considered topresent a low risk for recurrence are eligible.

b) Other active malignancy requiring concurrent intervention

c) Prior organ allograft

d) Previous treatment:

-   -   i) Prior anti-cancer treatments are permitted (i.e,        chemotherapy, radiotherapy, hormonal, or immunotherapy)    -   ii) Toxicity (except for alopecia) related to prior anti-cancer        therapy and/or surgery must either have resolved, returned to        baseline or Grade 1 or have been deemed irreversible    -   iii) For cytotoxic agents at least 4 weeks must have elapsed        between the last dose of prior to anti-cancer therapy and        initiation of study therapy    -   iv) For non-cytotoxic agents at least 4 weeks or 5 half-lives        (whichever is shorter) must have elapsed from last dose of prior        anti-cancer therapy and the initiation of study therapy.

e) Prior therapy with anti-OX40 antibody

f) Subjects with active, known, or suspected autoimmune disease areexcluded. Subjects with vitiligo, type 1 diabetes mellitus, residualhypothyroidism due to autoimmune condition only requiring hormonereplacement, euthyroid subjects with a history of Grave's disease(subjects with suspected autoimmune thyroid disorders must be negativefor thyroglobulin and thyroid peroxidase antibodies and thyroidstimulating immunoglobulin prior to first dose of study drug), psoriasisnot requiring systemic treatment, or conditions not expected to recur inthe absence of an external trigger are permitted to enroll. Subjectswith well controlled asthma and/or mild allergic rhinitis (seasonalallergies) are eligible.

g) Subjects with history of life-threatening toxicity related to priorimmune therapy (e.g., anti-CTLA-4 or anti-PD-1/PD-L1 treatment or anyother antibody or drug specifically targeting T-cell co-stimulation orimmune checkpoint pathways) except those that are unlikely to re-occurwith standard countermeasures (e.g., hormone replacement after adrenalcrisis)

h) Subjects with interstitial lung disease that is symptomatic or thatmay interfere with the detection or management of suspected drug-relatedpulmonary toxicity

i) Chronic obstructive pulmonary disease requiring recurrent steroidbursts or chronic steroids at doses greater than 10 mg/day of prednisoneor the equivalent

j) Subjects with a condition requiring systemic treatment with eithercorticosteroids (>10 mg daily prednisone equivalents) or otherimmunosuppressive medications within 14 days of study drugadministration except for adrenal replacement steroid doses >10 mg dailyprednisone equivalent in the absence of active autoimmune disease. Note:Treatment with a short course of steroids (<5 days) up to 7 days priorto initiating study drug is permitted.

k) Uncontrolled or significant cardiovascular disease, including but notlimited to any of the following:

-   -   i) Myocardial infarction or stroke/transient ischemic attack        within the past 6 months    -   ii) Uncontrolled angina within the past 3 months    -   iii) Any history of clinically significant arrhythmias (such as        ventricular tachycardia, ventricular fibrillation, or torsades        de pointes)    -   iv) History of other clinically significant heart disease (e.g.,        cardiomyopathy, congestive heart failure with New York Heart        Association functional classification III-IV, pericarditis,        significant pericardial effusion)    -   v) Cardiovascular disease-related requirement for daily        supplemental oxygen therapy    -   vi) QT interval corrected for heart rate using Fridericia's        formula (QTcF) prolongation >480 msec

l) History of any chronic hepatitis as evidenced by the following:

-   -   i) Positive test for hepatitis B surface antigen    -   ii) Positive test for qualitative hepatitis C viral load (by        PCR) Note: Subjects with positive hepatitis C antibody and        negative quantitative hepatitis C by PCR are eligible. History        of resolved hepatitis A virus infection is not an exclusion        criterion. Additional testing or substitute testing per        institutional guidelines to rule out infection is permitted.

m) Evidence of active infection that requires systemic antibacterial,antiviral, or antifungal therapy ≤7 days prior to initiation of studydrug therapy (does not apply to viral infections that are presumed to beassociated with the underlying tumor type required for study entry)

n) Known history of testing positive for HIV or known acquiredimmunodeficiency syndrome.

o) Evidence or history of active or latent tuberculosis infectionincluding PPD recently converted to positive; chest x-ray with evidenceof infectious infiltrate; recent unexplained changes in fever/chillpatterns.

p) Any major surgery within 4 weeks of study drug administration.Subjects must have recovered from the effects of major surgery orsignificant traumatic injury at least 14 days before the first dose ofstudy drug.

q) Use of non-oncology vaccines containing live virus for prevention ofinfectious diseases within 4 weeks prior to study drug. The use ofinactivated seasonal influenza vaccines, e.g., Fluzone®, is permitted.

r) Use of pRBC or platelet transfusion within 2 weeks prior to the firstdose of study drug

s) A known or underlying medical or psychiatric condition and/or socialreason that could make the administration of study drug hazardous to thesubjects or could adversely affect the ability of the subject to complywith or tolerate the study.

3) Allergies and Adverse Drug Reaction

a) History of allergy to nivolumab or ipilimumab (Parts 2 and 3 only,respectively)

b) History of any significant drug allergy (such as anaphylaxis orhepatotoxicity) to prior anti-cancer immune modulating therapies (e.g.,checkpoint inhibitors, T-cell co-stimulatory antibodies)

Study Assessments:

Physical examinations, vital sign measurements, 12-leadelectrocardiograms (ECGs), and clinical laboratory evaluations areperformed at selected times throughout the dosing interval. Subjects areclosely monitored for AEs throughout the study.

-   -   Safety Assessments: AEs are assessed during the study and for        100 days after the last treatment. AEs are evaluated according        to NCI CTCAE v4.03. Subjects are followed until all        treatment-related AEs have recovered to baseline or are deemed        irreversible.    -   Efficacy Assessments: Disease assessment with CT and/or MRI as        appropriate are performed at baseline and every 8 weeks (±1        week) for q2w dosing regimens and every 9 weeks (±1 week) for        q3w dosing regimens, then every 12 weeks during the treatment        and response follow-up phases until discontinuation of treatment        or withdrawal from study. Tumor assessments at other time points        are performed if there are concerns about tumor progression.        Assessment of tumor response is made according to RECIST v1.1        for subjects with malignant tumors.    -   Pharmacokinetic and Immunogenicity Assessments: Samples for PK        and immunogenicity assessments are collected for subjects        receiving OX40.21 alone or in combination with nivolumab or        ipilimumab. The PK of OX40.21 is characterized by        non-compartmental analysis (NCA) method. Immunogenicity samples        are analyzed for anti-OX40.21 antibodies and/or anti-nivolumab        antibodies and/or anti-ipilimumab antibodies by validated        immunoassays.    -   Exploratory Biomarker Assessments: To explore potential        predictive markers for clinical response to OX40.21 in relation        to dose and PK, 3 types of specimens are obtained from all        subjects for biomarker testing: (i) whole blood, (ii)        serum/plasma, and (iii) tumor tissue.

Statistical Considerations Sample Size Determination Dose Escalation:

As a Phase 1 dose escalation trial, the sample size for each doseescalation cohort depends on observed toxicity and posterior inference.Approximately 30 subjects are treated during each dose escalation part(OX40.21 monotherapy [Part 1A], OX40.21 in combination with nivolumab[Part 2A], and OX40.21 in combination with ipilimumab [Part 3A]) for acombined total of about 90 subjects in Parts 1A, 2A, and 3A. Initially,approximately 3 subjects are treated at the starting dose levels ofOX40.21 or OX40.21 in combination with nivolumab or ipilimumab.Additional cohorts of approximately 3 evaluable subjects are treated atrecommended dose levels per BLRM (−Copula) recommendations during thedose escalation phase. At least 6 DLT-evaluable subjects are treated atthe MTD.

Dose Expansion:

In general terms, the expansion phase sizing is based on target responserates (target overall response rate) and the ability to identify asignal for such clinical response that is above the standard of care(historical overall response rate).

Approximately 12 subjects are treated in the Part 1B dose expansioncohort. Approximately 35 subjects are treated in the Part 2B doseexpansion cohort. Approximately 27 subjects are treated in the Part 2Cdose expansion cohort. Approximately 35 subjects are treated in the Part3B dose expansion cohort.

Endpoints Primary Endpoints

The assessment of safety is based on the incidence of AEs, serious AEs,AEs leading to discontinuation, and deaths. In addition, clinicallaboratory test abnormalities are examined.

Secondary Endpoints

Efficacy: The anti-tumor activity of OX40.21 alone and OX40.21 incombination with nivolumab or ipilimumab is measured by ORR, duration ofresponse, and progression free survival rate (PFSR) at 24 weeks based onRECIST v1.1. The above are determined based on tumor measurementsoccurring at baseline, every 8 weeks (±1 week) for q2w dosing regimensand every 9 weeks (±1 week) for q3w dosing regimens during the treatmentperiod, and every 3 months (12 weeks) during the survival follow-upperiod.

-   -   Best overall response (BOR) is assessed per RECIST 1.1 criteria.    -   ORR is the proportion of all treated subjects whose BOR is        either CR or PR.    -   Duration of response, computed for all treated subjects with a        BOR of CR or PR, is the time between the date of first response        and the date of disease progression or death, whichever occurs        first.    -   PFSR at 24 weeks is defined as the proportion of treated        subjects remaining progression free and surviving at 24 weeks.        The proportion is calculated by the Kaplan-Meier estimate, which        takes into account censored data.

Pharmacokinetics

Selected parameters, such as Cmax, Tmax, AUC(0-t), and AUC(TAU), areassessed in 2 cycles depending on the schedule for monotherapy or incombination with nivolumab or ipilimumab. Parameters such as Ctau, CLT,Css-avg, accumulation index (AI), and effective elimination half-life(T-HALFeff) are assessed in the second cycle when intensive PK iscollected.

Immunogenicity

The secondary objective of immunogenicity is assessed by the frequencyof positive ADA to OX40.21 or nivolumab or ipilimumab.

Exploratory Endpoints

Exploratory objectives related to OS are assessed by OS rate at acertain time point (e.g., 2 years). OS rate is the proportion ofsubjects alive at that time point. OS for a subject is defined the timefrom the date of first dose of study medication to the date of deathfrom any cause. Exploratory objectives related to biomarkers areassessed by the change from baseline or baseline level biomarkermeasurements in peripheral blood (e.g., soluble factors including, butnot limited to, cytokine and chemokines) or tumor tissue (e.g.,tumor-infiltrating lymphocytes).

For subjects with multiple ECG measurements, the following parametersare optionally assessed: changes in the ECG intervals QT, QTc, QRS, andP-R interval from baseline.

Analyses

Safety analyses: All recorded AEs are listed and tabulated by systemorgan class, preferred term, and treatment. Vital signs and clinicallaboratory test results are listed and summarized by treatment. Anysignificant physical examination findings and clinical laboratoryresults are also noted. ECG readings are evaluated, and abnormalities,if present, are noted.

Efficacy analyses: Listing of tumor measurements are provided by subjectand study day in each arm and dose level. Individual subject's BOR islisted based on RECIST 1.1. To describe the anti-tumor activity ofOX40.21 alone or in combination with nivolumab or ipilimumab, ORR iscalculated. ORR and corresponding 2-sided 95% CI by the Clopper-Pearsonmethod are provided by treatment and/or dose level and tumor type.Median duration of response and corresponding 2-sided 95% CI arereported by treatment and/or dose level and tumor type. Duration ofresponse is analyzed using the Kaplan-Meier method. In addition, PFSR,the probability of a subject remaining progression free or surviving to24 weeks, is estimated by the Kaplan-Meier methodology by treatment,tumor type, and dose level. The corresponding 95% CI is derived based onGreenwood formula. OS is plotted using the Kaplan-Meier method. MedianOS and corresponding 2-sided 95% CI are reported.

Pharmacokinetic analyses: All individual PK parameters are listed foreach analyte, including any exclusions and reasons for exclusion fromsummaries. Summary statistics are tabulated for each PK parameter bytreatment. Geometric means and coefficients of variation are presentedfor Cmax, AUC(0-t), AUC(TAU), Ctau, CLT, Cssavg, and AI. Medians andranges are presented for Tmax. Means and standard deviations arepresented for all other PK parameters (e.g., T-HALFeff).

OX40.21 dose dependency is assessed in dose escalation monotherapy. Todescribe the dependency on dose of OX40.21, scatter plots of Cmax,AUC(0-t), and AUC(TAU) versus dose are provided for each day measured.An exploratory assessment of dose proportionality based on a power modeland a CI around the power coefficient is performed. Nivolumab andipilimumab end of infusion and trough (Ctrough) concentrations andOX40.21 trough concentration are tabulated by treatment and study dayusing summary statistics. These data may also be pooled with otherdatasets for population PK analysis.

Immunogenicity analysis: All available immunogenicity data are providedby treatment, dose, and immunogenicity status. The frequency of subjectswith positive ADA assessment of OX40.21, nivolumab, and ipilimumab aredetermined.

Exploratory biomarker analyses: Summary statistics for biomarkers andtheir corresponding changes (or percent changes) from baseline aretabulated by planned study day and dose in each arm. The time course ofbiomarker measures are represented graphically. If there is indicationof meaningful pattern over time, further analysis (e.g., by linear mixedmodel) is performed to characterize the relationship. Methods such as,but not limited to, logistic regression are used to explore possibleassociations between biomarker measures from peripheral blood or tumorbiopsy and clinical outcomes.

TABLE 23 SUMMARY OF SEQUENCES SEQ ID Description Sequence   1 Human OX40MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVS precursorRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADA HSTLAKI   2Extracellular LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKdomain PCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPG of matureDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQ human OX40PTEAWPRTSQGPSTRPVEVPGGRAVAA   3 CynomolgusMCVGARRLGRGPCAALLLLGLGLSTTAKLHCVGDTYPSNDRCCQECRPGNGMVS OX40RCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVAAILGLGLALGLLGPLAMLLALLLLRRDQRLPPDAPKAPGGGSFRTPIQEEQADA HSALAKI   4Human OX40-L MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSTLQVSHRYPRIQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGFYLISLKGYFS QEVNISLHYQ KDEEPLFQLK KVRSVNSLMV ASLTYKDKVYLNVTTDNTSL DDFHVNGGEL ILIHQNPGEF CVL*   5 human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL constantTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT domainKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG   6 human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL constantTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT domainKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (allotypicTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR variant)VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG   7 human IgG1RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA kappa lightLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ chain GLSSPVTKSFNRGEC   8heavy chain LSPGK constant region alternative C-terminus   9 heavy chainLSPG constant region alternative C-terminus  10 Human IgG1RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS kappa lightGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV chain TKSFNRGECconstant region (CL)  11 3F4 VH CDR1 SYDVN  12 3F4 VH CDR2WMNPNSGNTGYAPKFQG  13 3F4 VH CDR3 IYSSSYNWFDP  14 3F4 VL CDR1RASQSVSSYLA  15 3F4 VL CDR2 DASNRAT  16 3F4 VL CDR3 QQRSNWPLT  17 3F4 VHQVQLVQSGAEVKKPGASVKVSCKASGNTFTSYDVNWVRQATGQGLEWMGWMNPNSGNTGYAPKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR IYSSSYNWFDPWGQGTLVTVSS 18 3F4 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GGGTKVEIK  1914B6 VH CDR1 SNWIG  20 14B6 VH CDR2 FIYPGDSDTRYSPSFQG  21 14B6 VH CDR3YGDDWYFDL  22 14B6 VL1 CDR1 RASQSVSSYLA  23 14B6 VL1 CDR2 DASNRAT  2414B6 VL1 CDR3 QQRGDWPIT  25 14B6 VL2 CDR1 RASQGISSWLA  26 14B6 VL2 CDR2AASSLQS  27 14B6 VL2 CDR3 QQYNSYPRIT  28 14B6 VHEVQLEQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGFIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDIAMYYCAR YGDDWYFDLWGRGTLVTVSS 29 14B6 VL1 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWFQQRPGQAPRLLIYDASNRATGIPARFSGSGSGTDFSLTISSLEPEDFAVYYCQQRGDWPITF GQGTRLEIK  30 14B6 VL2DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRIT FGQGTRLEIK  3123H3 VH CDR1 NYAMY  32 23H3 VH CDR2 AIGIGGDTFYTDSVKG  33 23H3 VH CDR3MGTGYFFDY  34 23H3 VL CDR1 RASQSVSSYLA  35 23H3 VL CDR2 DASNRAT  3623H3 VL CDR3 QQRSNWPLT  37 23H3 VHEVQLVQSGGGLVHPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDMAVYYCARM GTGYFFDYWGQGTLVTVSS 38 23H3 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GPGTKVDIK  396E1 VH CDR1 SFAMH  40 6E1 VH CDR2 VISYDGSIKYYTDSVKG  41 6E1 VH CDR3DGNYGSARYFQH  42 6E1 VL1 CDR1 RASQGISSWLA  43 6E1 VL1 CDR2 AASSLQS  446E1 VL1 CDR3 QQYNSYPRT  45 6E1 VL2 CDR1 RASQSVSSYLA  46 6E1 VL2 CDR2DASNRAT  47 6E1 VL2 CDR3 QQRSNWPYT  48 6E1 VHQVQLVESGGGWQPGRSLRLSCAASGFTFSSFAMHWVRQAPGKGLEWVTVISYDGSIKYYTDSVKGRFTFSRDNSKNTLYLQMNSLRAEDTAVYYCTRDGNYGSARYFQHWGQGTLVTVSS  49 6E1 VL1DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTF GQGTKVEIK  50 6E1 VL2EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPYTF GQGTKLEIK  5118E9 VH CDR1 SSAMH  52 18E9 VH CDR2 AIGTGGDTYYADSVKG  53 18E9 VH CDR3DFYDILTGIFDY  54 18E9 VL CDR1 RASQGISSWLA  55 18E9 VL CDR2 AASSLQS  5618E9 VL CDR3 QQANSFPST  57 18E9 VHEVQLVQSGGGLVHPGGSLRLSCAHSGFTFTSSAMHWVRQAPGKGLEWISAIGTGGDTYYADSVKGRFTISRDNAKNSLYLQINSLRAEDMAVYYCARD FYDILTGIFDYWGQGTLVTVSS 58 18E9 VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPSTF GQGTKVEIK  598B11 VH CDR1 SDAMY  60 8B11 VH CDR2 AIGIGGDTYYTDSVMG  61 8B11 VH CDR3LGMGYYFDY  62 8B11 VL CDR1 RASQSVSSYLA  63 8B11 VL CDR2 DASNRAT  648B11 VL CDR3 QQRSNWPPT  65 8B11 VHMEFVLSWVFLVAILKGVQCEIQLVQSGGGLVHPGGSLRLSCAGSGFTFSSDAMYWVRQAPGKGLEWVSAIGIGGDTYYTDSVMGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARLGMGYYFDYWGQGTLVTVSS  66 8B11 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF GQGTKVEIK  6720B3 VH CDR1 SYDMH  68 20B3 VH CDR2 VIGTAGDTYYPGSVKG  69 20B3 VH CDR3GGMGNYFDY  70 20B3 VL CDR1 RASQSVSSYLA  71 20B3 VL CDR2 DASNRAT  7220B3 VL CDR3 QQRSNWPLT  73 20B3 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQTTGKGLEWVSVIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARG GMGNYFDYWGQGTLVTVSS 74 20B3 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GGGTKVEIK  7514A2 VH CDR1 NYALH  76 14A2 VH CDR2 LISYDGSRKHYADSVKG  77 14A2 VH CDR3LTMVREGG  78 14A2 VL1 CDR1 RASQSVSSSYLA  79 14A2 VL1 CDR2 GASSRAT  8014A2 VL1 CDR3 QQYGSSPFT  81 14A2 VL2 CDR1 RVSQGISSYLN  82 14A2 VL2 CDR2SASNLQS  83 14A2 VL2 CDR3 QRTYNAPYT  84 14A2 VHQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS LTMVREGGQGTLVTVSS  8514A2 VL1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFT FGPGTKVDIK  8614A2 VL2 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQKPGKVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYGQRTYNAPYTF GGGTKVEIK  8720C1 VH CDR1 SYAMY  88 20C1 VH CDR2 AIDTDGGTFYADSVRG  89 20C1 VH CDR3LGEGYFFDY  90 20C1 VL CDR1 RASQSVSSYLA  91 20C1 VL CDR2 DASNRAT  9220C1 VL CDR3 QQRSNWPPT  93 20C1 VHEAQLVQSGGGLVHPGGSLRLSCADSGFTFSSYAMYWVRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNGLRAEDMAVYFCARL GEGYFFDYWGQGTLVTVSS 94 20C1 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF GGGTKVEIK  95OX40.6 heavy EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLE chainWVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  96 OX40.6 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL chainLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  97 OX40.7 heavyEVQLVQSGGGLVHPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLE chainWVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARYGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  98 OX40.7 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL chainLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  99 OX40.8 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 100 OX40.8 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 101 OX40.9 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 102 OX40.9 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 103 OX40.10 heavy chainQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREGGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 104 OX40.10 light chainEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 105 OX40.11 heavy chainQVQLVESGGGWQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREGGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 106 OX40.11 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 107 OX40.12 heavy chainQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 108 OX40.12 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 109 OX40.13 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 110 OX40.13 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 111 OX40.14 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 112 OX40.14 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 113 OX40.15 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 114 OX40.15 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 115 OX40.16 heavyEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE chainWVSAIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 116 OX40.16 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL chain(sharedLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS by OX40.20,NWPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN OX40.21,FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA OX40.22)DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 117 OX40.17 heavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQTTGKGLE chainWVSVIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARGGMGNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 118 OX40.17 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL chainLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 119 OX40.18 heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVNWVRQATGQGLE chainWMGWMNPNSGNTGYAPKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARIYSSSYNWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 120 OX40.18 lightEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL chainLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121 OX40.19 heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE chainWVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTLVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 122 OX40.19 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chainLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 123 OX40.20 heavyEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE chainWVSAIDTSGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG — OX40.20 light SEQ ID NO: 116 chain124 OX40.21 heavy EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE chainWVSAIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG — OX40.21 light SEQ ID NO: 116 chain125 OX40.22 heavy chain EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSAIDTSTGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG — OX40.22 light SEQ ID NO: 116 chain126 3F4 VH CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT (nucleotideCAGTGAAGGTCTCCTGCAAGGCTTCTGGAAACACCTTCACCAGTTATGA sequence)TGTCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACACAGGCTATGCACCGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAATATATAGCAGCTCGTACAACTGGTTCGACCCCTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCA 1273F4 VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 128 14B6 VHGAGGTGCAGCTGGAGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGTTCATCTATCCTGGTGACTCTGATACCAGGTACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTCAAGGCCTCGGACATCGCCATGTATTACTGTGCGAGATATGGGGATGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCA CTGTCTCCTCA 12914B6 VL1 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTTCCAACAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCTCTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTGGCGACTGGCCCATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 130 14B6 VL2GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 131 23H3 VHGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAACTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCCATTGGTATTGGTGGTGACACATTCTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTGCAAGAATGGGAACTGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 132 23H3 VLGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA 133 6E1 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTTTGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGACAGTTATTTCATATGATGGAAGCATTAAATACTACACAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAGATGGAAACTATGGTTCGGCGAGATACTTCCAGCACTGGGGCCAGGGCA CCCTGGTCACCGTCTCCTCA134 6E1 VL1 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 135 6E1 VL2GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 136 18E9 VHGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCATCCTGGGGGGTCCCTGAGACTCTCCTGTGCACACTCTGGATTCACCTTCACTAGCTCTGCTATGCACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAATGGATATCAGCTATTGGTACTGGTGGTGACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATAAACAGCCTGAGAGCCGAGGACATGGCTGTATATTACTGTGCAAGAGACTTTTACGATATTTTGACTGGTATCTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCA 13718E9 VL GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCTTCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 138 8B11 VHATGGAGTTTGTGCTGAGCTGGGTTTTCCTTGTTGCTATATTAAAAGGTGTCCAGTGTGAAATTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAGCGATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGGTATTGGTGGTGACACATACTATACAGACTCCGTGATGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTGCAAGGCTGGGGATGGGGTACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCA 1398B11 VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 140 20B3 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACGACATGCACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGGGGGGATGGGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 141 20B3 VLGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 142 14A2 VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCAGGGAACCCTGGTCACCGTCTCCT CA 143 14A2 VL1GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA 144 14A2 VL2GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGTGAGTCAGGGCATTAGCAGTTATTTAAATTGGTATCGGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATAGTGCATCCAATTTGCAATCTGGAGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACGGTCAACGGACTTACAATGCCCCTTACACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 145 20C1 VHGAGGCTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGACTCTGGATTCACCTTCAGTAGCTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGATACTGATGGTGGCACATTCTATGCAGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACGGCCTGAGAGCCGAGGACATGGCTGTGTATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 146 20C1 VLGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 147 OX40.6 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAACTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCCATTGGTATTGGTGGTGACACATTCTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAATGGGAACTGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGT 148 OX40.6 lightGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 149 OX40.7 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAACTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCCATTGGTATTGGTGGTGACACATTCTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGATATGGAACTGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGT 150 OX40.7 lightGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 151 OX40.8 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 152 OX40.8 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 153 OX40.9 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 154 OX40.9 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 155 OX40.10 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 156 OX40.10 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 157 OX40.11 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 158 OX40.11 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 159 OX40.12 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 160 OX40.12 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 161 OX40.13 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 162 OX40.13 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 163 OX40.14 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 164 OX40.14 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 165 OX40.15 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 166 OX40.15 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 167 OX40.16 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAGCTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGATACTGATGGTGGCACATTCTATGCAGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA 168 OX40.16GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG light chainAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT (shared byAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT OX40.20,GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG OX40.21,GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA OX40.22)TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG 169OX40.17 heavy GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACGACATGCACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGGGGGGATGGGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA 170 OX40.17 lightGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 171 OX40.18 heavyCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG chainCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATGTCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACACAGGCTATGCACCGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAATATATAGCAGCTCGTACAACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCCCCGGGTTGA 172OX40.18 light GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 173 OX40.19 heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA chainGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGTCTTACTCTGGTTCGGGAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGTAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 174 OX40.19 lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG chainGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 175 OX40.20 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAGCTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGATACTAGTGGTGGCACATTCTATGCAGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA — OX40.20 lightSEQ ID NO: 168 chain 176 OX40.21 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAGCTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGATACTGATGCTGGCACATTCTATGCAGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA — OX40.21 lightSEQ ID NO: 168 chain 177 OX40.22 heavyGAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG chainGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAGCTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGCTATTGATACTAGTACTGGCACATTCTATGCAGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA — OX40.22 lightSEQ ID NO: 168 chain 178 hOX40 epitope DVVSSKPCKPCTWCNLR 179hOX40 epitope DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK 180 peptide linkerPVGVV 181 sortase A LPXTG, wherein X is any amino acid recognition motif182 hOX40 epitope QNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR 183 hOX40 epitopePCKPCTWCNLR 184 hOX40 epitope QLCTATQDTVCR 185 hOX40 epitopeSQNTVCRPCGPGFYN 186 IgG1 C- VDKRV termianl C_(H)1 (same for IgG3(17-15-15- 15), igG3 (17- 15-15), IgG3 (17-15), IgG3 (15-15-15),IgG3 (15), and IgG4 187 IgG2 C- VDKTV terminal C_(H)1 188 IgG1 upperEPKSCDKTHT hinge 189 IgG3 (17-15- ELKTPLGDTTHT 15-15) upper hinge (samefor IgG3 (17- 15-15) and IgG3 (17-15)) 190 IgG3 (15-15- EPKS 15) upperhinge (same for IgG3(15)) 191 IgG4 upper ESKYGPP hinge 192 IgG1 middleCPPCP hinge 193 IgG2 middle CCVECPPCP hinge 194 IgG3 (17-15-CPRCP(EPKSCDTPPPCPRCP)₃ 15-15) middle hinge 195 IgG3 (17-15-CPRCP(EPKSCDTPPPCPRCP)₂ 15) middle hinge 196 IgG3 (17-15)CPRCP(EPKSCDTPPPCPRCP)₁ middle hinge 197 IgG3 (15-15-CDTPPPCPRCP(EPKSCDTPPPCPRCP)₂ 15) middle hinge 198 IgG3 (15) CDTPPPCPRCPmiddle hinge 199 IgG4 middle hinge CPSCP 200 IgG1 lower APELLGGhinge (same for IgG3 (17- 15-15-15), IgG3 (17-15- 15), IgG3(17-15), IgG3 (15-15-15), IgG3 (15), and IgG4) 201 IgG2 lower hingeAPPVAG 202 Wildtype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGhuman IgG1 CH1 VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 203Wildtype ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGhuman IgG2 CH1 VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV 204Wildtype PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN human IgG1AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT CH2 ISKAK 205 WildtypePSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN human IgG2AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT CH2 ISKTK 206 WildtypeGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN humanNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ IgG1 CH3 KSLSLSPG 207Wildtype GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN humanNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ IgG2 CH3 KSLSLSPGK 208Alternative ERKCCVECPPCPAPPVAG hinge 209 Alternative ERKSCVECPPCPAPPVAGhinge 210 Alternative ERKCSVECPPCPAPPVAG hinge 211 AlternativeERKXCVECPPCPAPPVAG hinge 212 Alternative ERKCXVECPPCPAPPVAG hinge 213Alternative ERKCCVECPPCPAPPVAGX hinge 214 AlternativeERKSCVECPPCPAPPVAGX hinge 215 Alternative ERKCSVECPPCPAPPVAGX hinge 216Alternative ERKXCVECPPCPAPPVAGX hinge 217 AlternativeERKCXVECPPCPAPPVAGX hinge 218 Alternative ERKCCVECPPCPAPELLGG hinge 219Alternative ERKSCVECPPCPAPELLGG hinge 220 AlternativeERKCCSVECPPCPAPELLGG hinge 221 Alternative ERKXCVECPPCPAPELLGG hinge 222Alternative ERKCXVECPPCPAPELLGG hinge 223 Alternative ERKCCVECPPCPAPELLGhinge 224 Alternative hinge ERKSCVECPPCPAPELLG 225 AlternativeERKCCSVECPPCPAPELLG hinge 226 Alternative ERKXCVECPPCPAPELLG hinge 227Alternative ERKCXVECPPCPAPELLG hinge 228 Alternative ERKCCVECPPCPAPhinge 229 Alternative hinge ERKSCVECPPCPAP 230 Alternative hingeERKCSVECPPCPAP 231 Alternative hinge ERKXCVECPPCPAP 232Alternative hinge ERKCXVECPPCPAP 233 Portion of hinge PVAG 234Portion of hinge ELLG 235 Portion of hinge ELLGG 236 Portion of hingeSCDKTHT 237 Portion of hinge CCVE 238 WT human IgG2 hingeERKCCVECPPCPAPPVAG 239 Human IgG2 hinge ERKSCVECPPCPAPPVAG with C219S240 IgG2/IgG1 hinge ERKCCVECPPCPAPELLGG 241 IgG2 (C219S)/IgG1 hingeERKSCVECPPCPAPELLGG 242 Wild type human EPKSCDKTHTCPPCPAPELLGGIgG1 hinge 243 Human IgG1 CH2 withPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN A330S/P331SAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAK 244IgG1-IgG2-IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 245 IgG1-IgG2-1gG1f2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 246 IgG1-IgG2CS-IgG1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 247 IgG1-IgG2CS-IgG1f2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 248 IgG2-IgG1fASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 249 IgG2-IgG1f2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 250 IgG2CS-IgG1fASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 251 IgG2CS-IgG1f2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 252 IgG1-IgG2-IgG1.1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRREMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 253 IgG1-IgG2CS-IgG1.1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 254 IgG2-IgG1.1fASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 255 IgG2CS-IgG1.1fASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 256 IgG1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 257 IgG1.1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRV7QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 258 IgG2.3ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 259 IgG2.5ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 260 IgG2.3G1-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 261 IgG2.5G1-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 262 IgG2.3G1-AYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 263 IgG2.5G1-AYASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 264 IgG2.3G1.1f-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 265 IgG2.5G1.1f-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 266 IgG2.5G1-V27ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 267IgG2.3-V13 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 268 IgG2.3-V14ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDOEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 269 IgG2.3-V15ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 270 IgG2.3-V16ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDOEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 271 IgG2.3-V17ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 272 IgG2.3-V18ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVBHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 273 IgG2.3-V19ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVRHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 274 IgG2.3G1-AY-V20ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 275 IgG2.3G1-AY-V21ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 276 IgG2.3G1-AY-V22ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 277 IgG2.3G1-AY-V23ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 278 IgG2.3G1-AY-V24ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDOEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPFPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 279 IgG2.3G1-AY-V25ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 280 IgG2.3G1-AY-V26ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPDLLGDtJSVFLFPPKPKDTLMISRTPEVTCVWDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 281 IgG2.3G1-AY-V28ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 282 OX40.6-Vh-hHC-IgG2.3EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 283OX40.8-Vh-hHC-IgG2.3 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALXSYDGSRKHYADSVKGRFSXSRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 284OX40.16-Vh-hHC-IgG2.3 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 285 OX40.6-Vh-hHC-EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS IgG2.3G1AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 286 OX40.8-Vh-hHC-QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA IgG2.3G1LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 287 OX40.16-Vh-hHC-EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS IgG2.3G1AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 288 OX40.6-Vh-hHC-EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS IgG2.3G1-V27AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 289 OX40.8-Vh-hHC-QVQLVESGGGWQPGRSIiRLSCAASGFTFSNYALHWVRQAPGKGLEWVA IgG2.3G1-V27LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 290 OX40.16-Vh-hHC-EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS IgG2.3G1-V27AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 291OX40.6-Vh-hHC-IgG2.5 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 292OX40.8-Vh-hHC-IgG2.5 QVQLVESGGGWQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 293OX40.16-Vh-hHC-IgG2.5 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 294OX40.21-Vh-hHC-IgG2.5 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 295 OX40.21-Vh-hHC-EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS IgG2.5G1AIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 296OX40.21-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSIgG2.5G1-V27 AIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 297 IgG2.3G1-V27ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 298 Human IgG1 CH2 withPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY A330S/P331SVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAK 299Heavy chain-nivolumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDT AVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 300 Light chain-nivolumabEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPR TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC301 Heavy chain variableQVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA region-nivolumabVIWYDGSKRYYADSVKGRFTISRDNS KNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSS 302Light chain variable EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYregion-nivolumab DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 303 HCDR1-nivolumab NSGMH 304 HCDR2-nivolumabVIWYDGSKRYYADSVKG 305 HCDR3-nivolumab NDDY 306 LCDR1-nivolumabRASQSVSSYLA 307 LCDR2-nivolumab DASNRAT 308 LCDR3-nivolumab QQSSNWPRT309 Heavy chain variableQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVT region-ipilimumabFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR (from WO01/014424)TGWLGPFDYWGQGTLVTVSS 310 Light chain variableEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI region-ipilimumabYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT (from WO01/014424)FGQGTKVEIK 311 HCDR1-ipilimumab SYTMH (from WO01/014424) 312HCDR2-ipilimumab FISYDGNNKYYADSVKG (from WO01/014424) 313HCDR3-ipilimumab TGWLGPFDY (from WO01/014424) 314 LCDR1-ipilimumabRASQSVGSSYLA (from WO01/014424) 315 LCDR2-ipilimumab GAFSRAT(from WO01/014424) 316 LCDR3-ipilimumab QQYGSSPWT (from WO01/014424) 317HCDR2 of OX40.21 AIDTDAGTFYADSVRG 318 VH of OX40.21EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV YFCARLGEGYFFDYWGQGTLVTVSSTable 23 provides the sequences of the mature variable regions and heavyand light chains and where indicated, sequences with signal peptides.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments disclosed herein. Such equivalents are intended to beencompassed by the following claims.

1-25. (canceled)
 26. A method of producing an antibody which binds tohuman OX40 (anti-OX40 antibody), comprising (a) culturing a cell whichcomprises a nucleic acid encoding a heavy chain variable region (VH)and/or a light chain variable region (VL) of the anti-OX40 antibody, and(b) recovering the anti-OX40 antibody that is produced.
 27. The methodof claim 26, wherein the VH and the VL of the anti-OX40 antibodycomprise the amino acid sequence set forth in (a) SEQ ID NOs: 318 and94, respectively; (b) SEQ ID NOs: 17 and 18, respectively; (c) SEQ IDNOs: 28 and 29, respectively; (d) SEQ ID NOs: 28 and 30, respectively;(e) SEQ ID NOs: 37 and 38, respectively; (f) SEQ ID NOs: 48 and 49,respectively; (g) SEQ ID NOs: 48 and 50, respectively; (h) SEQ ID NOs:57 and 58, respectively; (i) SEQ ID NOs: 65 and 66, respectively; (j)SEQ ID NOs: 73 and 74, respectively; (k) SEQ ID NOs: 84 and 85,respectively; (l) SEQ ID NOs: 84 and 86, respectively; or (m) SEQ IDNOs: 93 and
 94. 28. The method of claim 27, wherein the VH comprises theamino acid sequence set forth in SEQ ID NO: 318 and the VL comprises theamino acid sequence set forth in SEQ ID NO:
 94. 29. An immunoconjugatecomprising an antibody which binds to human OX40 (anti-OX40 antibody)linked to an agent, wherein the anti-OX40 antibody comprises a heavychain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3,wherein the heavy chain CDR1 comprises the amino acid sequence set forthin SEQ ID NO: 87, the heavy chain CDR2 comprises the amino acid sequenceset forth in SEQ ID NO: 317, the heavy chain CDR3 comprises the aminoacid sequence set forth in SEQ ID NO: 89, the light chain CDR1 comprisesthe amino acid sequence set forth in SEQ ID NO: 90, the light chain CDR2comprises the amino acid sequence set forth in SEQ ID NO: 91, and thelight chain CDR3 comprises the amino acid sequence set forth in SEQ IDNO:
 92. 30. The immunoconjugate of claim 29, wherein the anti-OX40antibody comprises a heavy chain variable region (VH) and a light chainvariable region (VL), wherein the VH comprises the amino acid sequenceset forth in SEQ ID NO: 318 and the VL comprises the amino acid sequenceset forth in SEQ ID NO:
 94. 31. The immunoconjugate of claim 29, whereinthe anti-OX40 antibody comprises a heavy chain and a light chain,wherein the heavy chain comprises the amino acid sequence set forth inSEQ ID NO: 124 and the light chain comprises the amino acid sequence setforth in SEQ ID NO:
 116. 32. A method of enhancing an anti-tumorimmunotherapy in a subject in need thereof, comprising administering tothe subject the anti-tumor immunotherapy in combination with an isolatedantibody which binds to human OX40 (“anti-OX40 antibody”), wherein theanti-OX40 antibody comprises a heavy chain CDR1, CDR2, and CDR3, and alight chain CDR1, CDR2, and CDR3, wherein: (a) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 87, 317, and 89, respectively, and the light chain CDR1, CDR2, andCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 90-92,respectively; (b) the heavy chain CDR1, CDR2, and CDR3 comprises theamino acid sequences set forth in SEQ ID NOs: 11-13, respectively, andthe light chain CDR1, CDR2, and CDR3 comprises the amino acid sequencesset forth in SEQ ID NOs: 14-16, respectively; (c) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 19-21, respectively, and the light chain CDR1, CDR2, and CDR3comprises the amino acid sequences set forth in SEQ ID NOs: 22-24,respectively; (d) the heavy chain CDR1, CDR2, and CDR3 comprises theamino acid sequences set forth in SEQ ID NOs: 19-21, respectively, andthe light chain CDR1, CDR2, and CDR3 comprises the amino acid sequencesset forth in SEQ ID NOs: 25-27, respectively; (e) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 31-33, respectively, and the light chain CDR1, CDR2, and CDR3comprises the amino acid sequences set forth in SEQ ID NOs: 34-36,respectively; (f) the heavy chain CDR1, CDR2, and CDR3 comprises theamino acid sequences set forth in SEQ ID NOs: 39-41, respectively, andthe light chain CDR1, CDR2, and CDR3 comprises the amino acid sequencesset forth in SEQ ID NOs: 42-44, respectively; (g) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 39-41, respectively, and the light chain CDR1, CDR2, and CDR3comprises the amino acid sequences set forth in SEQ ID NOs: 45-47,respectively; (h) the heavy chain CDR1, CDR2, and CDR3 comprises theamino acid sequences set forth in SEQ ID NOs: 51-53, respectively, andthe light chain CDR1, CDR2, and CDR3 comprises the amino acid sequencesset forth in SEQ ID NOs: 54-56, respectively; (i) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 59-61, respectively, and the light chain CDR1, CDR2, and CDR3comprises the amino acid sequences set forth in SEQ ID NOs: 62-64,respectively; (j) the heavy chain CDR1, CDR2, and CDR3 comprises theamino acid sequences set forth in SEQ ID NOs: 67-69, respectively, andthe light chain CDR1, CDR2, and CDR3 comprises the amino acid sequencesset forth in SEQ ID NOs: 70-72, respectively; (k) the heavy chain CDR1,CDR2, and CDR3 comprises the amino acid sequences set forth in SEQ IDNOs: 75-77, respectively, and the light chain CDR1, CDR2, and CDR3comprises the amino acid sequences set forth in SEQ ID NOs: 78-80,respectively, wherein, optionally, the Asp-Gly sequence in SEQ ID NO: 76is replaced an amino acid sequence that does not undergo isomerization;(l) the heavy chain CDR1, CDR2, and CDR3 comprises the amino acidsequences set forth in SEQ ID NOs: 75-77, respectively, and the lightchain CDR1, CDR2, and CDR3 comprises the amino acid sequences set forthin SEQ ID NOs: 81-83, respectively; or (m) the heavy chain CDR1, CDR2,and CDR3 comprises the amino acid sequences set forth in SEQ ID NOs:87-89, respectively, and the light chain CDR1, CDR2, and CDR3 comprisesthe amino acid sequences set forth in SEQ ID NOs: 90-92, respectively.33. The method of claim 32, wherein the anti-OX40 antibody comprises aheavy chain variable region (VH) and a light chain variable region (VL),wherein the VH and VL comprise the amino acid sequence set forth in: (a)SEQ ID NOs: 318 and 94, respectively; (b) SEQ ID NOs: 17 and 18,respectively; (c) SEQ ID NOs: 28 and 29, respectively; (d) SEQ ID NOs:28 and 30, respectively; (e) SEQ ID NOs: 37 and 38, respectively; (f)SEQ ID NOs: 48 and 49, respectively; (g) SEQ ID NOs: 48 and 50,respectively; (h) SEQ ID NOs: 57 and 58, respectively; (i) SEQ ID NOs:65 and 66, respectively; (j) SEQ ID NOs: 73 and 74, respectively; (k)SEQ ID NOs: 84 and 85, respectively; (l) SEQ ID NOs: 84 and 86,respectively; or (m) SEQ ID NOs: 93 and
 94. 34. The method of claim 32,wherein the anti-OX40 antibody comprises a heavy chain and a lightchain, wherein the heavy chain and the light chain comprise the aminoacid sequence set forth in: (a) SEQ ID NOs: 124 and 116, respectively;(b) SEQ ID NOs: 95 and 96, respectively; (c) SEQ ID NOs: 97 and 98,respectively; (d) SEQ ID NOs: 99 and 100, respectively; (e) SEQ ID NOs:101 and 102, respectively; (f) SEQ ID NOs: 103 and 104, respectively;(g) SEQ ID NOs: 105 and 106, respectively; (h) SEQ ID NOs: 107 and 108,respectively; (i) SEQ ID NOs: 109 and 110, respectively; (j) SEQ ID NOs:111 and 112, respectively; (k) SEQ ID NOs: 113 and 114, respectively;(l) SEQ ID NOs: 115 and 116, respectively; (m) SEQ ID NOs: 117 and 118,respectively; (n) SEQ ID NOs: 119 and 120, respectively; (o) SEQ ID NOs:121 and 122, respectively; (p) SEQ ID NOs: 123 and 116, respectively;and (q) SEQ ID NOs: 125 and 116, respectively.
 35. The method of claim32, wherein the anti-tumor immunotherapy comprises an immune checkpointinhibitor.
 36. The method of claim 35, wherein the immune checkpointinhibitor comprises an anti-PD-1 antibody.
 37. The method of claim 32,wherein the subject has a cancer.
 38. The method of claim 37, whereinthe cancer comprises a cervical cancer, colorectal cancer, bladdercancer, ovarian cancer, or combinations thereof.
 39. The method of claim32, comprising administering one or more additional therapeutic agentsto the subject.
 40. The method of claim 39, wherein the one or moreadditional therapeutic agents comprise an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody,anti-TGFβ antibody, or combinations thereof.
 41. The method of claim 26,wherein the anti-OX40 antibody is a human antibody.
 42. Theimmunoconjugate of claim 29, wherein the anti-OX40 antibody is a humanantibody.
 43. The method of claim 32, wherein the anti-OX40 antibody isa human antibody.
 44. A composition comprising the immunoconjugate ofclaim 29 and a carrier.
 45. The composition of claim 44, which comprisesone or more additional therapeutic agents.